To explore the expression spectra of apoptosis-related gene pnas-2 in normal tissues and acute leukemia (AL) patient tissues, the expressions of pnas-2 gene in tissues including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, lymph node, thymus, leukocyte, bone marrow and fetal liver were detected by Northern blot. The expressions of pnas-2 in samples including 44 de novo, 9 non-CR, 27 CR and 12 relapsed AL patients were measured by real-time RT-PCR and Northern blot, and the expression levels of pnas-2 in normal and tumor tissues from 31 patients with malignancies were also detected. The results showed that pnas-2 was not expressed in the most tissues except in placenta. The results of real-time PCR indicated that pnas-2 expressions in samples of de novo, non-CR and relapsed patients ware significantly higher than that in CR, tumor tissues and normal tissues. In serial monitoring of 7 AL patients, the expression level of pnas-2 was high at first visit examination, but remarkably decreased after remission, and the pnas-2 expression level increased again when relapsed. It is concluded that the pnas-2 is specifically up-regulated in acute leukemia patients, which might be an oncogene and participate in leukemogenesis.
Acute Disease ; Apoptosis ; genetics ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Biomarkers, Tumor ; genetics ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia ; pathology

OBJECTIVETo investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2) phosphorylation status in the regulation of ASPP2-p53 apoptotic pathway activity.

METHODSCells were individually transfected with green fluorescent protein (GFP)-encoding vector, constitutively non-phosphorylatable ASPP2 mutant-ASPP2 (Am)-encoding vector, and wild type ASPP2 (Aw)-encoding vector) plasmids, respectively, to make them overexpressing phosphorylated and non-phosphorylated ASPP2 proteins, respectively. Cell apoptosis was induced by oxaliplatin. The apoptosis rate of cells was determined by flow cytometry after staining with FITC-conjugated annexin V and PI. ASPP2 protein level and its phosphorylation status were observed by Western blot. The interaction between ASPP2 and p53 was observed by immunoprecipitation assay.

RESULTSOxaliplatin induced cell apoptosis and caused phosphorylation of ASPP2 at ser92/ser361 in the HCT116 cells. The apoptosis rate of Aw and Am plasmids-transfected cells were (3.8 ± 1.0)% and (3.9 ± 1.2)% respectively, statistically with a non-significant difference (P > 0.05) in comparison with that of the GFP plasmid-transfected cells [(4.0 ± 0.8)%]. After oxaliplatin treatment, the apoptosis rate of Aw plasmid-transfected cells was (46.7 ± 3.9)%, significantly higher than that of the Am and GFP plasmid-transfected cells [(40.1 ± 10.2)% and (37.1 ± 6.9)%, respectively, P < 0.05], however, there was no statistically significant difference (P > 0.05) between Am and GFP plasmid-transfected cells. These results indicate that phosphorylated ASPP2 promoted the oxaliplatin-induced apoptosis of HCT116 cells through a p53-dependent pathway. Phosphorylation status of ASPP2 influenced its binding activity to p53.

CONCLUSIONPhosphorylation status of ASPP2 modulates p53 apoptotic function in oxaliplatin-induced apoptosis of colorectal cancer HCT116 cells.

Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Colorectal Neoplasms ; metabolism ; HCT116 Cells ; Humans ; Organoplatinum Compounds ; Phosphorylation ; Tumor Suppressor Protein p53 ; genetics ; metabolism

Immediate early response gene X-1 (IEX-1) gene was discovered by Charles in 1993, which plays an important role in regulating apoptosis. Previous studies demonstrated that IEX-1 gene was expressed in a variety of human tissues and played an important role in regulating apoptosis and cell growth through a positive or negative way. It can inhibit cell growth and apoptosis. However, it can also promote cell apoptosis. There is evidence that IEX-1 is involved in viral infections and cardiovascular diseases and it is highly expressed in many tumor tissues. The regulatory mechanisms for IEX-1 under normal and pathophysiological conditions are complicated and remain largely unknown. In this paper, we reviewed the recent progress of IEX-1 in different disease models.
Apoptosis ; genetics ; Apoptosis Regulatory Proteins ; genetics ; physiology ; Cell Proliferation ; Humans ; Membrane Proteins ; genetics ; physiology ; Neoplasms ; genetics ; metabolism ; pathology ; Osteosarcoma ; metabolism ; pathology

OBJECTIVE: To explore the role of apoptosis stimulating proteins of p53 2 (ASPP2) and inhibitory member of the ASPP family (iASPP) in the occurrence of cervical cancer and the relation between these two proteins and p53. METHODS: We used immunohistochemical method to detect the expression of ASPP2, iASPP, p53 in 51 patients with early cervical cancer tissue, 53 patients with cervical intraepithelial neoplasia (CIN) II-III, 48 patients with CIN I and 45 patients with normal cervical tissue. The relation among ASPP2, iASPP and p53 was analyzed. RESULTS: When p53 was negative, the positive expression rate of ASPP2 in the cervical cancer group, the CIN II-III group, the CIN I group and the normal cervix group was gradually increased. There was significant difference between the CIN II-III group or the cervical cancer group and the normal cervix (P<0.05), but no statistical difference was found among the other groups (P>0.05). The positive expression rate of iASPP in the 4 groups gradually reduced, and the difference was significant difference between the cervical cancer group or the CIN II-III group and the normal cervix group (P<0.05). When the p53 was positive, there was no significant change in the expression of ASPP2 and iASPP in every group (P>0.05). CONCLUSION ASPP2 and iASPP may play an important role in the occurrence of early stage of cervical cancer by regulating the ability of wild type p53 in induction of apoptosis. ASPP2 and iASPP gene might be a potential molecular target for cervical carcinoma.
Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; Female ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Uterine Cervical Neoplasms ; genetics ; metabolism

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.
Apoptosis ; Apoptosis Regulatory Proteins ; chemistry ; genetics ; metabolism ; Autophagy ; Beclin-1 ; Caspase 3 ; metabolism ; HeLa Cells ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism

To investigate the effect of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells on apoptosis induced by chemotherapeutics. 
 Methods: A total of 30 osteosarcoma tissues of sensitive and resistant to chemotherapeutics were divided into a chemotherapy-sensitive group and a chemotherapy-resistant group. The mRNA expression levels of miR-30a and high mobility group protein A2 (HMGA2) in the chemotherapy-sensitive group and the chemotherapy-resistant group, and the mRNA expression levels of miR-30a in osteosarcoma U2-OS cells treated by cisplatin, doxorubicin and methotrexate at different concentrations were detected by real-time PCR. The expression levels of autophagy related protein Beclin 1, microtubule associated protein 1 light chain 3B (LC3B) and autophagy factor P62 were detected by Western blotting. The osteosarcoma U2-OS cells were transfected with miR-30a mimics and miR-30a inhibitors to construct a miR-30a high expression group, a miR-30a low expression group and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after treatment of cisplatin and doxorubicin in these 3 groups were detected by Western blotting; the level of autophagy was detected by monodansylcada (MDC) staining; the level of ROS was detected by dihydroethidium (DHE); the level of cell surviving rate was detected by cell counting kit-8 (CCK-8); the level of apoptosis was detected by annexin APC/PI double staining; the level of mitochondria oxidative damage was detected by mitochondrial membrane potential assay kit with JC-1 (JC-1 method). The interaction between miR-30a and HMGA2 was detected by dual luciferase reporter assay. The osteosarcoma U2-OS cells were transfected with HMGA2 mimics and HMGA2-shRNA to construct a high HMGA2 group, a low HMGA2 group, and a control group. The expression levels of Beclin 1, LC3B and P62 in osteosarcoma U2-OS cells after the treatment of cisplatin were detected by Western blotting.
 Results: The level of miR-30a in the chemotherapy-resistant tissues was significantly lower than that in the chemotherapy-sensitive tissues (P<0.05), and the expression of HMGA2 was opposite comparing to that of miR-30a (P<0.05). After the treatment by low concentration (5 μmol/L) of chemotherapeutics, the level of miR-30a was down-regulated in osteosarcoma U2-OS cells, accompanied with up-regulation of Beclin 1 and LC3B (P<0.01) and down-regulation of P62 (P<0.01). Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly decreased (P<0.05), and the expression level of P62 was significantly increased (P<0.05) in the miR-30a high expression group, which was opposite in the miR-30a low expression group. In the miR-30a high expression group treated by chemotherapeutics, the level of autophagy and the cell survival rate were lower than those in group with low expression of miR-30a, while the levels of ROS, the mitochondrial oxidative damage and the apoptosis were higher than those in group with low expression of miR-30a (all P<0.05). The targeting interaction between HMGA2 and miR-30a were verified by dual luciferase reporter assay. Compared with the control group, the expression levels of Beclin 1 and LC3B were significantly increased (P<0.05), and the expression level of P62 was significantly decreased (P<0.05) in the HMGA2 high expression group, which was opposite in the HMGA2 low expression group.
 Conclusion: Suppression of miR-30a/HMGA2-mediated autophagy in osteosarcoma cells is likely to enhance the therapeutic effect of chemotherapeutics.
Apoptosis ; Apoptosis Regulatory Proteins ; Autophagy ; Beclin-1 ; Bone Neoplasms ; Cell Line, Tumor ; HMGA2 Protein ; metabolism ; Humans ; MicroRNAs ; genetics ; Osteosarcoma

OBJECTIVETo study the effect of recombinant tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) on the expression of apoptosis and inflammatory related genes in human colon cancer cell line HCT-116.

METHODSAfter 24-hour treatment with recombinant human TRAIL protein, the expressions of apoptosis-related genes(Bcl-2, Bad, caspase-3, and caspase-8) and inflammation-related genes(TNF-α, IL-1β, and COX-2) were measured by real-time PCR and appropriate kits in HCT-116.

RESULTSAfter treatments of 10 μg/L and 100 μg/L recombinant human TRAIL proteins, the apoptotic rates of HCT-116 cells were 27.4% and 45.9%, respectively. Expressions of anti-apoptosis gene Bcl-2, pro-apoptosis gene Bad and apoptotic markers caspase-3 and caspase-8 were significantly up-regulated, which was more significant in the group of 100 μg/L treatment(P<0.05). Moreover, after TRAIL treatments, expressions of inflammation-related genes TNF-α, IL-1β, COX-2 were also dramatically increased, and 100 μg/L treatment group showed higher up-regulation(P<0.05).

CONCLUSIONSRecombinant TRAIL protein induces both apoptosis and inflammation of human colon cancer cells.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; HCT116 Cells ; Humans ; Inflammation ; Recombinant Proteins ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology

BACKGROUNDOvarian cancer is a leading gynecological malignancy. We investigated the prognostic value of programmed cell death 5 (PDCD5) in patients with ovarian cancer.

METHODSExpression levels of PDCD5 mRNA and protein were examined in six ovarian cancer cell lines (SKOV3, CAOV3, ES2, OV1, 3AO, and HOC1A) and one normal ovarian epithelial cell line (T29) using reverse transcription polymerase chain reaction, Western blotting, and flow cytometry. After inducing PDCD5 induction in SKOV3 cells or treating this cell line with taxol or doxorubicin (either alone or combined), apoptosis was measured by Annexin V-FITC/propidium iodide staining. Correlations between PDCD5 protein expression and pathological features, histological grade, FIGO stage, effective cytoreductive surgery, and serum cancer antigen-125 values were evaluated in patients with ovarian cancer.

RESULTSPDCD5 mRNA and protein expression were downregulated in ovarian cancer cells. Recombinant human PDCD5 increased doxorubicin-induced apoptosis in SKOV3 cells (15.96 ± 2.07%, vs. 3.17 ± 1.45% in controls). In patients with ovarian cancer, PDCD5 expression was inversely correlated with FIGO stage, pathological grade, and patient survival (P < 0.05, R = 0.7139 for survival).

CONCLUSIONSPDCD5 expression is negatively correlated with disease progression and stage in ovarian cancer. Therefore, measuring PDCD5 expression may be a good method of determining the prognosis of ovarian cancer patients.

Adult ; Aged ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Female ; Humans ; In Vitro Techniques ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Prognosis ; Young Adult

In order to further study the anti-tumor activity of bimS and the feasibility of using adenovirus vector for gene therapy,we constructed a recombinant adenovirus vector of pro-apoptotic factor bimS(pAdEasy-CMV-bimS). Human bimS gene was amplified from HL-60 leukemic cell by RT-PCR and it was identified by sequencing. Then bimS was cloned into the shuttle vector pAdTrack-CMV that carried a green fluorescence protein (GFP) gene to generate a recombinant plasmid pAdTrack-CMV-bimS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E. coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restriction endonuclease digestion. The recombinated adenovirus pAdEasy-CMV-bimS was transferred into HEK293 cell for packaging and amplification. The virus titre was determined and the insert of bimS gene was verified by PCR method. Finally, HEK293 cell was infected by recombinated adenovirus pAdEasy-CMV-bimS (MOI=100) and bimS protein was detected by western blot. GFP expression in transfected HEK293 cells could be observed at 48-72 hours. bimS gene was detected in cultural supernatant of infected HEK293 cell by PCR and there was typical cytopathic effect(CPE) in HEK293 cell 7 days after infection. Western blot showed bimS protein expression in infected HEK293 cells was markedly higher than that in uninfected HEK293 cells. The result indicated that human bimS recombinant adenovirus was constructed successfully, which could therefore provide a sound base for anti-tumor gene therapy with bimS in vivo and in vitro.
Adenoviridae ; genetics ; metabolism ; Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; metabolism ; HEK293 Cells ; HL-60 Cells ; Humans ; Neoplasms ; therapy ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo detect Beclin1 expression and explore its clinical significance in primary hepatocellular carcinoma (HCC).

METHODSBeclin1 expressions in 10 normal hepatic tissues, 30 hepatitis liver, 30 cirrhotic liver and 50 HCC tissues were detected by immunohistochemical staining.

RESULTSThe positivity rates of Beclin1 expression in the HCC, cirrhotic liver, hepatitis liver and normal liver tissues were 78.00% (39/50), 26.67% (8/30), 53.33% (16/30), and 10.00% (1/10), respectively, showing significant differences between them (chi(2)=28.31, P<0.05). Beclin1 expression was significantly higher in HCC tissues than in the cirrhotic, hepatitis and normal liver tissues (chi(2)=20.39, 5.31, and 14.41, respectively, P<0.05), and hepatitis tissues showed significantly higher Beclin1 expression than hepatic cirrhosis tissues and normal hepatic tissues (chi(2)=4.44 and 4.12, respectively, P<0.05).

CONCLUSIONThe abnormal expression of Beclin1 is closely associated with the pathogenesis and development of primary hepatocellular carcinoma, and may play an important role in this process.

Adult ; Aged ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Beclin-1 ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged