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 .

Prognostic values of apoptosis-stimulating P53-binding protein 1 and 2 and their relationships with clinical characteristics of esophageal squamous cell carcinoma patients:a retrospective study

Background: Esophageal squamous cell carcinoma(ESCC) is a leading cause of cancer?related death, and new prognostic biomarkers are urgently needed. Apoptosis?stimulating P53?binding protein 1(ASPP1) and 2(ASPP2) have been reported to play important roles in the development, progression, metastasis, and prognosis of cancers, but their roles in ESCC have not been elucidated. In this study, we examined the expression of ASPP1 and ASPP2 in ESCC to evaluate their prognostic values.Methods: The protein expression of ASPP1, ASPP2, and P53 in 175 specimens of ESCC was detected using immuno?histochemical staining; their expression in cancerous and noncancerous tissues was scored according to the stain?ing intensity and the percentage of stained cells. The associations of ASPP1, ASPP2, and P53 with clinicopathologic parameters, overall survival(OS), and disease?free survival(DFS) were analyzed.Results: The protein expression levels of ASPP2 and P53 were significantly higher in cancerous tissues than in paired noncancerous tissues(P < 0.001), whereas the expression levels of ASPP1 in the two groups were similar. In ESCCs, ASPP1 expression was significantly associated with histological differentiation(P = 0.002) and invasive depth(P = 0.014); ASPP2 expression was associated with age(P = 0.029) and histological differentiation(P < 0.001); and P53 expression was associated with age(P and P53 expression. Survival an= 0.021) and tumor size(P alysis revealed that high AS= 0.040). No correlations were found between ASPP1, ASPP2,PP2 expression was significantly associated with increased 5?year OS(P = 0.001) and DFS rates(P ate of ESCC patients(= 0.010) and that high P53 expression was significantly associated with a reduced 5?year DFS rP atio(HR): 0.541, 9= 0.015). Multivariate Cox analysis indicated that ASPP2 was an inde?pendent predictor of OS [hazard r5% confidence interval(CI) 0.363–0.804] and DFS(HR: 0.599, 95% CI 0.404–0.888) of ESCC patients and that P53 was an independent predictor of DFS(HR: 2.161, 95% CI 1.100–4.245).Conclusions: ASPP1 might be involved in the progression of ESCC, and ASPP2 was a potential prognostic biomarker of ESCC and should be evaluated in future studies.

Effects of nanometer particles and water decoction of the Chinese medicine mixture of pinellia ternate and scorpion on P53 protein contents and apoptosis in the cerebral cortex and hippocampus of epileptic rats

BACKGROUND： Water decoction of the Chinese traditional medicine mixture of pinellia ternate and scorpion is an effective treatment for epilepsy. OBJECTIVE： To compare nanometer particles and effects of water decoction of Chinese traditional medicine mixture on P53 protein levels and apoptosis in the cerebral cortex and hippocampus of epileptic rats. DESIGN, TIME AND SETTING： This randomized, controlled molecular biology study was performed at the Key Laboratory of Child Neural Rehabilitation of Jiamusi University from October to December 2007. MATERIALS： Forty healthy male Wistar rats were used in this study. Convulsion rat models were established by intraperitoneal infusion of 35 mg/kg pentylenetetrazol, once a day, for 28 successive days. The Chinese traditional medicine mixture, comprising pinellia ternate, scorpion, centipede, bupleurum, peach pit and glycyrrhiza, was purchased from Beijing Tongrentang, China. The mixture was made into nanometer particles and water decoction. The apoptosis determination kit and P53 immunohistochemistry kit were bought from Boster, China. METHODS： Forty Wistar rats were randomly divided into four groups of ten rats per group, control, nanometer particle, water decoction and epileptic model groups. Rats in the nanometer particle and water decoction groups were respectively treated with 300 mg/kg Chinese herb nanometer particle suspension and water decoction by gavage, once a day, for 28 days. Rats in the epileptic model group were fed an equal volume of saline by gavage. Rats in the control group were only administered with the same volume of saline by gavage. MAIN OUTCOME MEASURES： Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling （TUNEL） and immunohistochemistry were used to respectively detect neuronal apoptosis and P53 protein expression in the rat brain cortex and hippocampus at 28 days following administration. RESULTS： The number of apoptotic neurons was lower in the nanometer particle and water decoction groups compared with the epileptic model group. The number of apoptotic cells and P53 protein expression in the rat cerebral cortex and hippocampus was greater in the epileptic model group compared with the control group （P 〈 0.05）, but lower in the nanometer particle and water decoction groups compared with the epileptic model group （P 〈 0.05）, and lower in the nanometer particle group compared with the water decoction group （P 〈 0.05）. CONCLUSION： Chinese traditional medicine mixture decreases neuronal apoptosis and P53 protein expression in epileptic rats. The effect of nanometer particles is significant compared with water decoction.

Apoptosis and P53 protein expression after rat spinal cord injury

objective: To determine the DNA damage, neuronal and glial cells apoptosis and expression of P53 protein in the rat spinal cord after compression injury. Methods: Terminal deoxynucleotidyl transferase . mediated dUPT nick end labeling (TUNEL), DNA gel electrophoresis and immunohistochemistry techniques were used to detect DNA fragmentation in the injured rat spinal cord. Results: The apoptosic cells and P53 protein presented at 4 h after spinal cord injury with a maximum presence at 24 h in the injuried T8.9 DNA fragmentation presented typical ladder pattern on agarose gel at 24 h. Conclusion: There are lots of neuronal and glial cells apoptosis with DNA damage after SCl. The P53 protein may play an important role in induc tion of neuronal and glial cells to apoptosis.

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.

Oncoprotein expression and inhibition of apoptosis during colorectal tumorigenesis

AIMS To study bcl-2 and P53 protein expression and inhibition of apoptosis during colorectal tumorigenesis. METHODS Expression of bcl -2 and p53 in 45 colorectal ade- nomas and 61 colorectal carcinomas was detected by immunohis- tochemical staining. RESULTS The bcl-2 and P53 protein expression was uniformly negative in normal mucosa,whereas bcl-2 and p53 positive rates were significantly higher in adenoma and carcinoma than in nor- reals(P<0.01 ).The area with strong bcl-2 expression was of- ten the area with severely dysplasia.In colorectal adenoma,ex- pression of p53 increased with the increasing size and dysplasia, in adenomas≥20 mm being higher than adenomas<10 mm(77, 8% vs 35.0%,P<0.05).p53 was relevant to differentiation and Duke's staging.A significant inverse correlation was found between bcl-2 and p53 in immunostaining in the adenomas,but not in the carcinomas.Furthermore,carcinomas with a high per- centage of bcl-2 positive cells were significantly more likely to have low rates of apoptosis. CONCLUSIONS These results suggest that bcl-2 gene appears to be an early event in colorectal tumorigenesis that can inhibit apoptosis,p53 expression plays an important role in the develop- ment and malignant change of colorectal adenoma,bcl-2 and p53 may be used as a good marker relating to cell apoptosis.

Effects of Selenium Dioxide on Apoptosis, Bcl-2 and P53 Expression, Intracellular Reactive Oxygen Species and Calcium Level in Three Human Lung Cancer Cell Lines

Objective: To evaluate the anti-tumor effects of SeO2 and its mechanisms on three human lung cancer cell lines. Methods: Three lung cancer cells A549, GLC-82 and PG were treated with 3-30 μmol/L SeO2. Flow cytometry was used to detect apoptosis, and analyze the changes of expression of p53 and Bcl-2, as well as ROS and Ca2+ level within cells. Results:SeO2 markedly inhibited cell proliferation and viability, and prompted apoptosis after 48 h treatment. SeO2 at 10 μmol/L induced 47.8% apoptosis in A549 cells, 40.8% in GLC-82 cells, 18.2% in PG cells. SeO2 at 30 μmol/L induced 37.8% apoposis in PG cells,but did not increase apoptotic raes in other two cells. SeO2 could down-regulate the mean fluorescent intensity of Bcl-2 from 65.8 to 9.6 in A549, but not in GLC-82 and in PG cells, up-regulate wild type p53 level in all three cells. SeO2 decreased the ROS and Ca2+ level markedly within three tested cells. Conclusion: SeO2 showed anti-tumor effect via apoptosis pathway in three lung cancer cell lines. The decrease of ROS and Ca2+ level within cells as well as regulation of Bcl-2 and p53 expression may play important roles in above apoptotic procedure.

Relationship between therapeutic efficacy of arterial infusion chemotherapy and expression of P-glycoprotein and p53 protein in advanced hepatocellular carcinoma

AIM： To investigate the relationship between the chemotherapeutic drug efficacy and the expression of P-glycoprotein （PGP） and p53 protein in advanced hepatocellular carcinoma （HCC）. METHODS： The study was conducted on 41 patients with advanced HCC who were treated by repeated arterial infusion chemotherapy. Biopsy specimens from the tumor were collected before the start of treatment in all the patients, and the specimens were stored frozen until immunohistochemical staining, which was performed after the start of treatment, to detect PGP and p53 protein expressions. Twenty of the fortyone patients were treated with an anthracycline drug （epirubicin hydrochloride; anthracycline group）, and the remaining 21 were treated with a non-anthracycline drug （mitoxantrone hydrochloride in 11 patients and carboplatin in 10 patients; non-anthracycline group）. The relationship between the chemotherapeutic efficacy and the results of immunostaining were compared between the two groups. RESULTS： Before the start of the treatment, PGPpositive rate was 90.2% （strongly-positive, 36.6%） and p53 protein-positive rate was 34.1% （strongly-positive, 19.5%）. In the anthracycline group, the response rate was 40.0%. The number of patients showing poor response to the treatment was significantly larger in the patients with strongly positive PGP expression （P= 0.005）, and their prognoses were poor （P= 0.001）. in the nonanthracycline group, the response rate was 42.9%,and there was no significant relationship between the chemotherapeutic drug efficacy and the PGP or p53 protein expression. When only the data from the 11 patients treated with anthraquinone drug, mitoxantrone, were analyzed, however, the number of patients who showed poor response to treatment was significantly higher among the p53-positive patients （P= 0.012）, irrespective of the survival outcome. CONCLUSION： The chemotherapeutic efficacy with an anthracycline drug for advanced HCC can be predicted by immunohistochemical analysis of PGP expression. Similarly, immunostaining to evaluate p53 protein may be useful to predict the response in patients treated with an anthraquinone drug.

Saikosaponin D inhibits proliferation and induces apoptosis via C/EBPβ-p53 signal pathway in human hepatoma HepG2 cells

Objective To investigate the anticancer effects and detailed mechanisms of Saikosaponin D(SSD)in human hepatoma HepG2 cells.Methods Cell proliferation and apoptosis were tested by MTT assay and Annexin-V/PI assay respectively.The expressions of CCAAT enhancer binding protein β(C/EBPβ)and p53 were detected by RT-PCR and Western blotting.Results SSD inhibited cell proliferation in a dose-dependent manner and induced apoptosis at the concentration of 5.0 mg/L.SSD significantly increased the mRNA and protein levels of C/EBPβ and p53 in a dose-dependent manner.Conclusion SSD exerts its anticancer effect by inhibiting cell proliferation and inducing apoptosis partly through C/EBPβ-p53 signal pathway in HepG2 cells.

Killing of p53-deficient hepatoma cells by parvovirus H-1 and chemotherapeutics requires promyelocytic leukemia protein

AIM: To evaluate the synergistic targeting and killing of human hepatocellular carcinoma (HCC) cells lacking p53 by the oncolytic autonomous parvovirus (PV) H-1 and chemotherapeutic agents and its dependence on functional promyelocytic leukemia protein (PML). METHODS: The role of p53 and PML in regulating cy-totoxicity and gene transfer mediated by wild-type (wt) PV H-1 were explored in two pairs of isogenic human hepatoma cell lines with different p53 status. Further-more,H-1 PV infection was combined with cytostatic drug treatment. RESULTS: While the HCC cells with different p53 status studied were all susceptible to H-1 PV-induced apoptosis,the cytotoxicity of H-1 PV was morepronounced in p53-negative than in p53-positive cells. Apoptosis rates in p53-negative cell lines treated by genotoxic drugs were further enhanced by a treatment with H-1 PV. In flow cytometric analyses,H-1 PV infection resulted in a reduction of the mitochondrial transmembrane potential. In addition,H-1 PV cells showed a significant increase in PML expression. Knocking down PML expression resulted in a striking reduction of the level of H-1 PV infected tumor cell death. CONCLUSION: H-1 PV is a suitable agent to circumvent the resistance of p53-negative HCC cells to genotoxic agents,and it enhances the apoptotic process which is dependent on functional PML. Thus,H-1 PV and its oncolytic vector derivatives may be considered as therapeutic options for HCC,particularly for p53-negative tumors.

Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

Relationship between ras p53 gene RNA and protein expression and HCC metastasis

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THE STUDY ON RELATIONSHIP BETWEEN CIGARETTE SMOKING AND THE p53 PROTEIN AND P21 PROTEIN EXPRESSION IN NON-SMALL LUNG CANCER

This paper discusses the relationship between cigarette smoking and the p53 protein and P21 protein expression by the immunohistochemical analysis in 93 cases with lung cancer in which squamous cell carcinoma accounted for 45 cases, adenocarcinoma 48 cases. The results showed that positive proportion of p53 protein expression was 74.20% (28 of 37 squamous cell carcinoma, 21 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 38.46% (3 of 8 squamous cell carcinoma, 7 of 18 adenocarcinomas) in nonsmoking group with lung cancers. The difference was statistically significant. Odds ratio was 4.14 and confidence limits for OR was 1.42-12.52. A dose-related presents in the p53 protein expression for the smoking amount and smoking years. The positive proportion of P21 protein expression was 79.31% (21 of 28 squamous cell carcinoma, 25 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 82.75% (10 of 11 squamous, 14 of 18 adenocarcinomas) in nonsmoking group with lung cancers, the difference was not statistically significant. But their positive proportion of P21 protein expression were very high in both groups. It was indicated that no relationship between cigarette smoking and the P21 protein expression. We suggest that the p53 gene could be a common target of tobacco-associated carcinogenesis in lung cancer.

Expression of p53,Bax and Bcl-2 proteins in hepatocytes in non-alcoholic fatty liver disease

AIM: To analyze the protein expression essential for apoptosis in liver steatosis. METHODS: The expression of proapoptotic proteins p53, Bax, and antiapoptotic Bcl-2 in hepatocytes with steatosis (SH) and without steatosis (NSH) was evaluated in 84 patients at various stages of non-alcoholic fatty liver disease (NAFLD). RESULTS: Immunohistochemical staining of liver tissue showed the activation of p53 protein in SH and NSH with increased liver steatosis, diminished Bcl-2 and slightly decreased Bax protein. Positive correlation was found between the stage of liver steatosis with p53 expression in SH (r = 0.54, P < 0.01) and NSH (r = 0.49, P < 0.01). The antiapoptotic protein Bcl-2 was diminished together with the advancement of liver steatosis, especially in non-steatosed hepatocytes (r =0.43, P < 001). CONCLUSION: Apoptosis is one of the most important mechanisms leading to hepatocyte elimination in NAFLD. The intensification of inflammation in NAFLD induces proapoptotic protein p53 with the inhibition of antiapoptotic Bcl-2.

Induction of antiproliferative effect by diosgenin through activation of p53, release of apoptosis-inducing factor (AIF) and modulation of caspase-3 activity in different human cancer cells

Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenininduced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.

Effect of HCV NS_3 protein on P53 protein expression in hepatocarcinogenesis

AIM To investigate hepatocarcinogenesis by detecting the effect of HCV NS 3 protein on P53 protein expression in hepatocellular carcinoma (HCC) and pericarcinomatous liver tissue (PCLT). METHODS The expression of HCV NS 3 and P53 protein was detected with immunohistochemical technique (SP method) in specimens of HCC and PCLT from 47 patients with negative HBV. RESULTS The positive rate of HCV NS 3 protein was lower in HCC (62%) than in PCLT (83%) ( P <0 025). The better differentiaton of cancer cells, the stronger expression of HCV NS 3 protein ( P <0 025). The positive rate of P53 protein in HCC (81%) was higher than in PCLT (47%) ( P <0 025). The worse differentiaton of cancer cells, the stronger expression of P53 protein ( P <0 05). The P53 protein expression was not correlated with the HCV NS 3 protein expression in HCC ( P >0 5), whereas their expression was closely related to PCLT ( P <0 01), and the expression rate of P53 protein in the cases of positive HCV NS 3 protein was higher than that in the cases of negative HCV NS 3 protein. CONCLUSION HCV NS 3 protein may exert its hepatocarcinogenic effect in early stage on host cells by endogenous pathway which may bring about mutation of p53 gene and transformation of hepatocytes.

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

Recent studies indicate that cell-cycle checkpoints are tightly correlated with the regulation of apoptosis, in which p53 plays an important role. Our present works show that the expression of E6/E7 oncogenes of human papillomavirus in HeLa cells is inhibited in the presence of anti-tumor reagent tripchlorolide (TC), which results in the up-regulation of p53 in HeLa cells. Interestingly, under the same TC-treatment, the cells at the early S-phase are more susceptible to apoptosis than those at the middle S-phase although p53 protein is stabilized to the same level in both situations. Significant difference is exhibited between the two specified expression profiles. Further analysis demonstrates that anti-apoptotic gene survivin is up-regulated by p53 in the TC-treated middle-S cells, whereas it is down-regulated by p53 in the TC-treated early-S cells. Taken together, the present study indicates that the differential p53-regulated expres- sion of survivin at different stages of the cell cycle results in different cellular outputs under the same apoptosis-inducer.

Effect of Boschniakia rossica on expression of GSTP,p53 and p21^(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats

AIM To investigate the effect of Boschniakiarossica（BR）extract on expression of GST-P,p53 and p21rasproteins in early stage of chemicalhepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS The expression of tumor marker-placental form glutathione S-transferase（GST-P）,p53 and p21rasproteins were investigated byimmunohistochemical techniques and ABCmethod.Anti-inflammatory activities of BR werestudied by xylene and croton oil-induced mouseear edema,carrageenin,histamine and hotscald-induced rat pow edema,adjuvant-inducedrat arthritis and cotton pellet-induced mousegranuloma formation methods.RESULTS The 500 mg/kg of BR-H2O extractfractionated from BR-Methanol extract hadinhibitory effect on the formation of DEN-inducedGST-P-positive foci in rat liver（GST-P stainingwas 78% positive in DEN+AAF group vs 20%positive in DEN+AAF+BR group,P【0.05）andthe expression of mutant p53 and p21rasproteinwas lower than that of hepatic preneoplasticlesions（33% and 22% positive respectively inDEN+AAF group vs negative in DEN+AAF+BRgroup）.Both CH2Cl2 and H2O extracts from BRhad anti-inflamatory effect in xylene and crotonoil-induced mouse ear edema（inhibitory rateswere 26%-29% and 35%-59%,respectively）. BR-H2O extract exhibited inhibitory effect incarrageenin,histamine and hot scald-inducedhind paw edema and adjuvant-induced arthritis inrats and cotton pellet-induced granulomaformation in mice.CONCLUSION BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis. Both CH2CI2 and H2O extracts from BR exerted anti-inflammatory effect in rats and mice.

Glycyrrhizic acid attenuates CCl_4-induced hepatocyte apoptosis in rats via a p53-mediated pathway

AIM: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apo-ptosis in rats via a p53-dependent mitochondrial path-way. METHODS: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group,rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were de-termined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis. RESULTS: After 8 wk of treatment, GA significantly re-duced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P<0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P<0.05), attenuated the changes in liver his-topathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P<0.05) in CCl4 -treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7.87% ± 0.66% to 3.68% ± 0.32%, P<0.05) compared with the CCl4 group. GA also decreased the expression level of cleaved caspase-3 compared to the CCl4 group. TU-NEL assay indicated that GA significantly diminished the number of TUNEL-positive cells compared with the CCl4 group (P<0.05). GA treatment clearly decreased the level of p53 (P<0.05) detected by immunohis-tochemistry and Western blotting analysis. Compared with the CCl4 group, we also found that GA reduced the Bax/Bcl-2 ratio (P<0.05), the expression of cleaved caspase-3 (P<0.05), cleaved caspase-9 (P<0.05), and inhibited cytochrome C and second mitochondria-derived activator of caspases (Smac) release from mito-chondria to cytoplasm, i.e. , GA reduced the expressionlevel of Smac, which inhibited c-IAP1 activity (P<0.05), ultimately inhibiting the activity of caspase-3, according to Western blotting analysis. As a result, GA suppressed activation of the caspase cascades and prevented he-patocyte apoptosis.

Blocking NF-kB nuclear translocation leads to p53-related autophagy activation and cell apoptosis

AIM: To investigate the anti-tumor effects of nuclear factor-κB (NF-κB) inhibitor SN50 and related mechanisms of SGC7901 human gastric carcinoma cells. METHODS: MTT assay was used to determine the cytotoxic effects of SN50 in gastric cancer cell line SGC7901. Hoechst 33258 staining was used to detect apoptosis morphological changes after SN50 treatment. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after SN50 treatment.Immunofluorescence staining was used to detect the expression of light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Western blotting analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p53 upregulated modulator of apoptosis (PUMA), damage-regulated autophagy modulator (DRAM), LC3 and Beclin 1. We detected the effects of p53-mediated autophagy activation on the apoptosis of SGC7901 cells with the p53 inhibitor pifithrin-α. RESULTS: The viability of SGC7901 cells was inhibited after SN50 treatment. Inductions in the expression of apoptotic protein p53 and PUMA as well as autophagic protein DRAM, LC3 and Beclin 1 were detected with Western blotting analysis. SN50-treated cells exhibited punctuate microtubule-associated protein 1 LC3 in immunoreactivity and MDC-labeled vesicles increased after treatment of SN50 by MDC staining. Collapse of mitochondrial membrane potential Δψ were detected for 6 to 24 h after SN50 treatment. SN50-induced increases in PUMA, DRAM, LC3 and Beclin 1 and cell death were blocked by the p53 specific inhibitor pifithrin-α. CONCLUSION: The anti-tumor activity of NF-κB inhibitors is associated with p53-mediated activation of autophagy.