Smac Gene Research Articles

Tumor suppressor p73 induces apoptosis of murine peritoneal cell after exposure to hydatid cyst antigens; a possibly survival mechanism of cystic echinococcosis in vivo mice model.

Cystic echinococcosis (CE) is a life-threatening helminthic disease caused by the Echinococcus granulosus sensulato complex. Previous evidence indicates that the host's innate immune responses against CE can combat and regulate the growth rate and mortality of hydatid cyst in the host's internal organs. However, the survival mechanisms of CE are not yet fully elucidated in the human body. In the present study, the apoptotic effects of fertile and infertile hydatid fluid (HF) were tested on murine peritoneal cells in vivo mice model. Mice were divided into five groups including; control group, fertile HF-treated peritoneal cells, infertile HF-treated peritoneal cells, protoscolices (PSCs)-treated peritoneal cells and HF+PSCs-treated peritoneal cells group. Mice groups were intraperitoneally inoculated with PBS, HF, and/or PSCs. Afterwards, peritoneal cells were isolated and mRNA expression of STAT3, caspase-3, p73 and Smac genes were evaluated by quantitative Real-time PCR. After 48 hours of exposure, the protein levels of Smac and STAT3 was determined by western blotting technique. After 6 hours of exposure, Caspase-3 activity was also measured by fluorometric assay. The intracellular reactive oxygen species (ROS) production was examined in all groups. The mRNA expression levels of p73, caspase-3 and also Caspase-3 activity in HF+PSCs-treated peritoneal cells were higher than in the test and control groups (Pv<0.05), while the mRNA expression level of anti-apoptotic STAT3 and Smac genes in HF+PSC-treated peritoneal cells were lower than in the other groups (Pv<0.05). As well, the level of intracellular ROS in the fertile HCF-treated peritoneal cells, infertile HCF-treated peritoneal cells, PSC-treated peritoneal cells and HF+PSC-treated peritoneal cells groups were significantly higher than in the control group (Pv<0.05).Current findings indicates that oxidative stress and p73 can trigger the apoptosis of murine peritoneal cells through modulator of HF-treated PSCs that is likely one of the hydatid cyst survival mechanisms in vivo mice model.

Scrophularıa oxysepala inhibit oral cancer cell line OECM-1 through induction of apoptosis

Oral cancer is a deadly malignancy that indicated high resistance to chemotherapeutic agents. Natural compounds derived from herbal medicine are many popular, and commonly used for treatment of several human disorders and malignancies in the world. In this study we aimed to investigate the anticancer activity of Scrophularıa oxysepala essential oil on oral cancer cell line OECM-1 and underlying mechanisms. The essential oil of S. oxysepala prepared by Clevenger instrument from dried leaves of plant. Cytotoxic effects of S. oxysepala essential oil was investigated by Tetrazolium staining on OECM-1 oral cancer cells. Moreover, the effects of S. oxysepala essential oil was investigated on apoptosis rate in cancer cells. The apoptosis-related genes (BAX, BCL2, SMAC, SURVIVIN) expression in treated cancer cells were evaluated by quantitative Real-Time PCR method. Our results indicated a significant cytotoxic effect of S. oxysepala essential oil against oral cancer cells in a time- and concentration-dependent manner. We observed several morphological changes in treated oral cancer cells by S. oxysepala essential oil that indicate cancer cell death and apoptosis. In addition, S. oxysepala essential oil induced apoptosis rate in treated cancer cells through regulation of BAX, BCL2, SMAC, and SURVIVIN genes expression. In general, our study demonstrated a significant cytotoxic effects of S. oxysepala essential oil against oral cancer cells, which may be due to induction of apoptosis through regulation of apoptosis-related genes expression.

Melatonin increases the anticancer potential of doxorubicin in Caco-2 colorectal cancer cells.

Colorectal cancer (CC) is an important human malignancy with high cancer related death worldwide. The chemotherapy using doxorubicin hydrochloride is one of the most common cancer therapeutic methods. However, drug resistance lowers the treatment efficacy in CC patients. The combination therapies seem to be more promising by taking the advantage of synergistic effects. The present study aimed to evaluate a new strategy to enhance the anticancer activity of doxorubicin in Caco-2 CC cell line by co-administration of melatonin. The effects of doxorubicin, melatonin, and their combinations (Dox-Mel) were investigated on the proliferation and viability, morphological alterations, and tumor spheroid formation. Flow cytometry was employed to compare the apoptotic situation of the cells in study groups. Changes in metastatic potential of the cells were assessed by wound healing assay and trans-well migration assays. Moreover, expression of BAX, SMAC, BCL-2, SURVIVIN, MMP-2, and MMP-9 genes were evaluated by quantitative real time PCR and western blotting. Our study showed that doxorubicin, melatonin, and Dox-Mel significantly decreased the proliferation and viability, tumor spheroid formation, invasion, and migration. Furthermore, the changes were in a concentration and time dependent manner. There was an increase in apoptosis rate in the treatment groups. Expression of genes involved in apoptosis and cell motility were altered significantly. It was observed that anticancer activity of Dox-Mel combination was significantly more than doxorubicin and melatonin treatments alone. We showed an enhanced apoptotic and anticancer activity of doxorubicin and melatonin combination chemotherapy on CC cell line than doxorubicin or melatonin treatments alone. This combination could promote the treatment efficiency and alleviate the un-intended side effects by lowering the dose of doxorubicin prescription.

Anti-Cancer Effects of Probiotic Lactobacillus acidophilus for Colorectal Cancer Cell Line Caco-2 through Apoptosis Induction

Background: Colorectal cancer is one of the most common cancers worldwide. Probiotics are useful and non-pathogenic microorganisms in the gastrointestinal tract, which can show anticancer activity through the induction of apoptosis. This study aimed to evaluate the antiproliferative effects of Lactobacillus acidophilus probiotic on the Caco-2 colorectal cancer cell line. Methods: The supernatant (secreted metabolites) and bacterial extract of L. acidophilus probiotics were prepared and used as an anti-proliferative agent on the colorectal cancer cell line, Caco-2 in vitro. The effects of supernatant and extract of L. acidophilus were evaluated on the viability and proliferation of cancer cells using MTT assay. Moreover, morphological alterations of cancer cells treated with supernatant and extract of L. acidophilus were evaluated by an inverted phase contrast microscope. The mRNA expression levels of apoptosis-related genes (SURVIVIN and SMAC) in treated cancer cells and untreated controls were evaluated using the Real-Time PCR method. Results: The results showed that the supernatant and extract of L. acidophilus inhibited the viability and proliferation of cancer cells in a dose and time-dependent manner. Moreover, various morphological alterations were observed in the treated cancer cells, which are indicators of apoptosis induction. The mRNA expression of SURVIVIN and SMAC genes were significantly up-regulated and downregulated in the treated cancer cells, respectively. Conclusion: The results of the present study suggested that the supernatant and extract of L.acidophilus could inhibit the viability and proliferation of colorectal cancer cell line, Caco-2through induction of apoptosis, increase the survival rate of colon cancer patients.

Smac Gene Delivery by the Glycol Chitosan with Low Molecular Weight Polyethylenimine Induces Apoptosis of Cancer Cells for Combination Therapy with Etoposide

We developed polymer-based nanoparticle carriers for gene delivery, glycol chitosan (GC)-methyl acrylate-low molecular weight polyethylenimine (800Da PEI), GMP, and investigated their ability to deliver second mitochondria-activation of caspase (Smac) gene into cancer cell lines. We analyzed cytotoxicity level and gene transfection efficiency in GBL-14, U373-MG, MDA-MB-231, and dermal fibroblasts (HDF) cell lines. GMP exhibited significantly lower cytotoxicity level, as compared with PEI25KD. Also, it showed high transfection efficiency in these cell lines. We treated these cells with a complex of GMP and Smac, and investigated their facilitated cellular uptake by flow cytometry. Also, analyses using cell cycle distribution, Annexin V staining, JC-1, and caspase-3 activity, indicated that apoptosis was induced by GMP/FLAG-Smac combined with etoposide in MDA-MB-231 cell line. This study demonstrates that combining the GMP/FLAG-Smac complex with chemotherapeutic agents could potentially be an effective approach for cancer therapy.

Gemcitabine combined with an engineered oncolytic vaccinia virus exhibits a synergistic suppressive effect on the tumor growth of pancreatic cancer

Pancreatic cancer (PC) is a lethal solid malignancy with resistance to traditional chemotherapy. Recently, considerable studies have demonstrated the ubiquitous antitumor properties of gene therapy mediated by the oncolytic vaccinia virus. The second mitochondrial-derived activator of caspase (Smac) has been identified as an innovative tumor suppressor that augments the chemosensitivity of cancer cells. However, the therapeutic value of oncolytic vaccinia virus (oVV)-mediated Smac gene transfer in pancreatic cancer is yet to be elucidated. In the present study, oncolytic vaccinia virus expressing Smac (second mitochondrial-derived activator of caspase) (oVV-Smac) was used to examine its beneficial value when used alone or with gemcitabine in pancreatic cancer in vitro and in vivo. The expression of Smac was evaluated by western blot analysis and quantitative polymerase chain reaction, oVV-Smac cytotoxicity by MTT assay, and apoptosis by flow cytometry and western blot analysis. Furthermore, the inhibitory effect of oVV-Smac combined with gemcitabine was also evaluated. The results indicated that oVV-Smac achieved high levels of Smac, greater cytotoxicity, and potentiated apoptosis. Moreover, co-treatment with oVV-Smac and gemcitabine resulted in a synergistic effect in vitro and in vivo. Therefore, our findings advance oVV-Smac as a potential therapeutic candidate in pancreatic cancer and indicated the synergistic effects of co-treatment with oVV-Smac and gemcitabine.

Molecular mechanism of anti-cancerous potential of Morin extracted from mulberry in Hela cells

The flavonoids are of great interest due to their antioxidant and anti-cancerous potential. The present study investigated the Morin extracted from mulberry leaves and its anti-cancerous mechanism via cell inhibition, cell cycle analysis, apoptosis, mRNA expression and antioxidant mechanism through reactive oxygen species generation. Morin inhibited the proliferation of HeLa cells at IC50 of 214.28 μM and led to morphological changes, followed by induction of cell cycle arrest in G2/M-phase and ultimately resulted into apoptosis. Morin-induced G2/M-phase arrest was accompanied by the increase in mRNA expression of p53, p21 and Wee 1 genes and decreased levels of CDK1, Cdc25c, Survivin, cyclin B1and CHK2. Morin-induced apoptosis was regulated through multiple pathways, including intrinsic and extrinsic pathway. The underlying mechanisms consisted of increased mRNA expression of Bax, Bad, cytochrome c, Apaf-1, caspases-9, DR3, DR5, FasL, FADD, caspases-10, PARP, PI3K, AKT, mTOR, P70S6K and Smac genes as well as decreased expression of Bcl-2, Bcl-xL, AMPK, cIAP-1, cIAP-2, PKCε and NF-kβ. In addition, Morin treatment resulted in the generation of intracellular ROS, which play an essential role in the induction of apoptosis. The present study recommends the use of Morin in the development of functional foods with anti-cancerous potential in the future.

ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model

BackgroundParasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown. The rodent malaria parasites may be useful to model interactions of parasite species, which lack the var genes with their respective hosts, as other multigene families are shared between the species. The role of the endothelial receptors ICAM-1 and CD36 in cytoadherence and in the development of pathology was investigated in a Plasmodium chabaudi infection in C57BL/6 mice lacking these receptors. The schizont membrane-associated cytoadherence (SMAC) protein of Plasmodium berghei has been shown to exhibit reduced CD36-associated cytoadherence in P. berghei ANKA-infected mice.MethodsParasite tissue sequestration and the development of acute stage pathology in P. chabaudi infections of mice lacking CD36 or ICAM-1, their respective wild type controls, and in infections with mutant P. chabaudi parasites lacking the smac gene were compared. Peripheral blood parasitaemia, red blood cell numbers and weight change were monitored throughout the courses of infection. Imaging of bioluminescent parasites in isolated tissues (spleen, lungs, liver, kidney and gut) was used to measure tissue parasite load.ResultsThis study shows that neither the lack of CD36 nor the deletion of the smac gene from P. chabaudi significantly impacted on acute-stage pathology or parasite sequestration. By contrast, in the absence of ICAM-1, infected animals experience less anaemia and weight loss, reduced parasite accumulation in both spleen and liver and higher peripheral blood parasitaemia during acute stage malaria. The reduction in parasite tissue sequestration in infections of ICAM-1 null mice is maintained after mosquito transmission.ConclusionsThese results indicate that ICAM-1-mediated cytoadherence is important in the P. chabaudi model of malaria and suggest that for rodent malarias, as for P. falciparum, there may be multiple host and parasite molecules involved in sequestration.

Construction of Smac siRNA lentiviral vector and its silencing effect on Smac gene in human LECs

Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step. Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line(HLE-B3) and establish low-expressed Smac HLE-B3 line. Methods Based on the genebank and our previous study, siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GV118 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR. Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp, and viral titer was 3.0×108 TU/ml.At a MOI of 100, the infecting efficiency of the vector on HLE-B3 cells was about 82% and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)%, (92.330±6.320)% and (32.540±4.221)% in the negative control group, siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group, respectively, showing a significant difference among the three groups(F=32.871, P<0.01), and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P=0.000). However, no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535). Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs. Key words: Mitochondrial proteins; Apoptosis; RNA, small interfering; Genetic vectors; Lentivirus/genetics; Gene transfer techniques; Humans; Lens epithelial cells

Effects of hypoxia/radiation dual-sensitive promoter-Smac plasmid combined with X-rays irradiation on proliferation of hypoxic A549 cells

Objective To construct recombinant expression vector of Smac gene mediated by hypoxia/radiation dual-sensitive promoter (HRE/Egrl),and observe the expression characteristics in A549 cells under normoxic and hypoxic conditions after transfection,and the inhibitory effects on proliferation under hypoxic condition.Methods The plasmid pcDNA3.1-Egrl-Smac and pcDNA3.1-HRE/Egrl-Smac were successfully constructed,and transfected into A549 cells.The expression levels of mRNA and protein were detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting,respectively.Cell proliferation changes were measured by methyl thiazol tetrazolium (MTT) as-say.Results Under normoxic condition,as compared with control group,Smac mRNA was significantly increased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy (P ＜ 0.05).Hypoxia could significantly enhance the effects (P ＜ 0.05).At the same time,Smac protein had similar expression characteristics.Under normoxic and hypoxic conditions,at the same time point,as compared with control group,proliferation was significantly decreased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy (P ＜ 0.05),and proliferation in each group under hypoxic condition was more lower than that in each group under normoxic condition (P ＜ 0.05).Conclusion Both hypoxia and radiation could enhance the Smac expression in A549 cells,and combination of hypoxia and 2 Gy irradiation could significantly inhibit proliferation of A549 cells. Key words: X-rays ; Hypoxia; Lung adenocarcinoma; Proliferation

Effect of arctigenin on apoptosis of mouse erythroleukemia FBL-3 cells and its mechanism

Objective To investigate the effect of arctigenin on the apoptosis of FBL-3 cells and its mechanisms. Methods The mouse erythroleukemia FBL-3 cells were taken as subjects. The untreated FBL-3 parental cells were taken as the control group, and the FBL-3 cells treated with 20 mg/L arctigenin for 24 h were taken as the experimental group. The effects of arctigenin on the apoptosis of the mouse erythroleukemia FBL-3 cells were determined by agarose gel electrophoresis and flow cytometry assay. The changes of apoptosis-related genes were analyzed by reverse transcriptase-polymerase chain reaction(RT-PCR). Results The agarose gel electrophoresis results revealed that the DNA ladder was displayed in the experimental group compared to the control group. The flow cytometry data demonstrated that the apoptosis number of FBL-3 cells in the experimental group was markedly increased compared to that in the control group (t=60.681, P=0.000). The RT-PCR assay results suggested that the expressions of Bcl-2 and IAP-1 gene were down-regulated in the experimental group compared to the control group (t=14.732, 29.702, all P=0.000), while the expressions of Bax and Smac gene were up-regulated(t=6.721, 8.499; P=0.003, 0.001), but the expression of Bcl-XL did not change(t=0.209, P=0.844). Conclusions Arctigenin can induce the apoptosis of the mouse erythrolenkemia FBL-3 cells. Apoptosis of the mouse erythrolenkemia FBL-3 cells induced by arctigenin may be correlated to the down-regulation of Bcl-2 and IAP-1 and up-regulation of Bax as well as Smac. Key words: Arctigenin; Leukemia; Apoptosis; Anti-apoptosis gene; Pro-apoptosis gene

Effect of Smac on TRAIL-induced apoptosis of prostate cancer cell line PC-3 and the molecular mechanism

The effect of Smac gene on the TRAIL-induced apoptosis of the prostate cancer cell line PC-3 and the molecular mechanism were investigated. The Smac gene was transfected into PC-3 cells under the induction of liposome. The intrinsic Smac gene expression was detected by Western blotting. After treatment with TRAIL as an apoptosis inducer, in vitro cell growth activity was assayed by MTT colorimetry. The apoptosis rate of PC-3 cells was determined by annexin V-FITC and propidium iodide staining flow cytometry. The expression of cellular XIAP and caspase-3 genes was examined by Western blotting. Smac-transfected cells (PC-3/Smac group) had significantly increased Smac protein level as compared with PC-3 controls (P<0.01). After induction with 100-200 ng/mL TRAIL for 12-36 h, cellular proliferation rate in PC-3/Smac group was significantly lower than in PC-3 controls (P<0.05). After induction with 100 ng/mL TRAIL for 24 h, the apoptosis rate in PC-3/Smac group was significantly enhanced as compared with that of PC-3 controls (P<0.05). Accordingly, the XIAP expression level was down-regulated significantly (P<0.05) and caspase-3 subunit P20 was up-regulated significantly (P<0.05). It is suggested that the over-expression of cellular Smac can inhibit inhibitor of apoptosis proteins (IAPs), enhance caspases activity and the apoptosis rate of PC-3 cells induced by TRAIL, which may provide a useful experimental basis for prostate cancer therapy.

Regulation of wild type PTEN gene on Survivin, Xiap and Smac in chronic leukemia cells

To explore the effects of tumor-suppressing gene wild type PTEN on the cell proliferation, apoptosis and the possible regulations of apoptosis-related molecules Survivin, Xiap and Smac gene in human chronic myeloid leukemia (CML) and cell line K562 cells. (1) The recombinant adenovirus containing green fluorescent protein (GFP) and PTEN (Ad-PTEN-GFP) or empty vector (Ad-GFP) was transfected into K562 cells. The growth of K562 cells was observed by MTT assay while cell cycle and apoptotic rate were assessed by flow cytometry (FCM). PTEN, Survivin, Xiap and Smac mRNA levels were detected by real-time fluorescent relative-quantification reverse transcriptional PCR (FQ-PCR) while PTEN protein levels analyzed by Western blot. (2) The expression levels of PTEN, Survivin, Xiap and Smac mRNA were detected in 10 chronic myelogenous leukemia (CML) patients in chronic phase (CML-CP), 10 CML patients in blast crises (CML-BC) and 10 normal control marrow mononuclear cells (MMNC). The growth of K562 cells was suppressed markedly. And the maximal growth inhibition rate was 38.6% after the transfection of PTEN. Survivin, Xiap, Smac mRNA expression levels were down-regulated by around 6.14, 7.44 and 2.95 folds respectively (0.0700 ± 0.0059, 0.0089 ± 0.0006, 0.0600 ± 0.0039 vs 0.4370 ± 0.0790, 0.0661 ± 0.0072, 0.1580 ± 0.0078 vs 0.4530 ± 0.0810, 0.0700 ± 0.0079, 0.1770 ± 0.0085, all P < 0.01). The mRNA expression level of PTEN in CML-BC patients was lower than that in CML-CP patients and normal control. But Survivin, Xiap, Smac mRNA expression levels were higher in CML-BC patients than those in CML-CP and normal control. The over-expression of PTEN gene may inhibit the proliferation of K562 cells and promote cell apoptosis via the regulation of Survivin, Xiap and Smac genes.

Par-4 gene silence inhibited hydrogen peroxide-induced apoptosis in alveolar epithelial cells

Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide), hydrogen peroxide and Par-4-siRNA-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA), Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot.Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was (29.7 ± 2.3) %, which was significantly lower than that of hydrogen peroxide-treated group [(54.2 ± 4.1)%, q= 8.91, P ＜ 0.01)]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity. Key words: Par-4; Small RNA interference; Hydrogen peroxide; Alveolar epithelial cells; Apoptosis

Survivin and Smac Gene Expressions but not Livin Are Predictors of Prognosis in Non-small Cell Lung Cancer Patients Treated with Adjuvant Chemotherapy Following Surgery

Survivin and livin, which are members of the inhibitor of apoptosis protein family, regulate both programmed cell death and proliferation. Second mitochondria-derived activator of caspase is thought to regulate apoptosis by antagonizing inhibitor of apoptosis protein. These gene expressions are regarded as prognostic markers in some malignancies. However, result in previous studies of the association of these gene expressions with prognosis of patients with non-small cell lung cancer remains contradictory. Survivin, livin and second mitochondria-derived activator of caspase mRNA was detected by semi-quantitative reverse transcriptase-polymerase chain reaction in surgical resected tumor specimen from 66 non-small cell lung patients who received adjuvant platinum-based chemotherapy. Results showed that patients with survivin high expression had significantly shorter tumor-free survival (P = 0.012) and overall survival (P = 0.007) than those with survivin low expression. There was a significant association of second mitochondria-derived activator of caspase high expression in non-small cell lung cancer tissue with longer tumor-free survival (P = 0.021) and overall survival (P = 0.0013). However, livin mRNA expression level had no impact on the tumor-free survival and overall survival of the patients. In multivariate analyses, survivin mRNA high expression (P = 0.033 and P = 0.024) and advanced pathologic stage (P = 0.009 and P = 0.008) were the factors which independently predicted a worse tumor-free survival and overall survival. Our data suggest that assessment of survivin and second mitochondria-derived activator of caspase mRNA expression may be useful for predicting survival in non-small cell lung cancer patients receiving platinum-based chemotherapy after surgical resection and can provide valuable information for deciding better therapy strategy.

Lead Affects Apoptosis and Related Gene XIAP and Smac Expression in the Hippocampus of Developing Rats

Lead (Pb) exposure poses devastating effects on central nervous system development of children. To replicate aspects of this neurotoxicity, we examined the effect of lead on the expression of apoptosis and of apoptosis-related genes, XIAP (X chromosome-linked inhibitor of apoptosis protein) and Smac (second mitochondrial activator of caspase), in the hippocampus of developing rats. A total of 48 rats (30-day old) were randomly divided into four groups for intragastrical perfusion of lead acetate [Pb(Ac)2]: untreated, low (2 mg/kg/d), medium (20 mg/kg/d), and high (200 mg/kg/d) dose groups. Pb content was determined in blood, and the apoptosis indexes and XIAP and Smac gene expression were analyzed in the hippocampus. There was a significant difference in apoptosis indexes (AI) between the exposed and control groups (p < 0.01). AI was highest in the high exposure group. XIAP gene expression was reduced in the exposed groups and the expression was negatively correlated with blood lead levels (BLLs) (p < 0.05). But the four groups did not differ in the expression of Smac (p > 0.05). Our data indicate that exposure to Pb(Ac)2 caused a dose-dependent and significant increase of apoptosis in the hippocampus of developing rats through depressing the expression of the XIAP but not the Smac genes.

The expression and significance of Smac, Livin and Caspase-3 gene in osteosarcoma

Objective To explore the expression of Samc, Livin and Caspase-3 genes in osteosar-coma,and its effect on biological behaviors of osteosarcoma. Methods Using immunohistochemistry meth-od (SABC), the expression of Smac, Livin and Caspase-3 protein was detected in 46 cases of osteosarco-mas. The relationship between the expression of Smac and Livin genes with clinicopathological parameters of osteosarcomas was analyzed. Results There was expression of Smac, Livin and Caspase-3 genes in 29 cases (63.0%), 30 cases (65.2%) and 32 cases (72.4%) in osteosarcomas, respectively. The expres-sion rate of Smac and Livin had no relationship with histologic staging and WHO types of osteosarcoma, but correlated with metastasis (P<0.05). Smac had a positive correlation with Livin expression (r=0.639,P<0.01), but had no relationship with Caspase-3 expression in osteosarcomas. Conclusion Apoptosis correlated factors, Smac, Livin and Caspase-3 were all highly expressed in osteosarcoma, and maybe they arc involved in the occurrence and development of osteosarcoma by modulating apoptosis. Key words: Osteosarcoma; Caspase-3; Apoptosis

Multimolecular complex of Par-4 and E2F1 binding to Smac promoter contributes to glutamate-induced apoptosis in human- bone mesenchymal stem cells

Neural cells undergo glutamate-induced apoptosis in ischaemic brain tissue, in which prostate apoptosis response-4 gene (Par-4) is involved. Human-bone mesenchymal stem cells can be utilized as an effective therapy for ischemic brain injury. In this study, we found that glutamate could induce apoptosis in human-bone mesenchymal stem cells, accompanied by increased expression of Par-4 gene and Smac release from mitochondria. Repressing Par-4 expression attenuated the glutamate-induced apoptosis. Both Par-4 protein and E2F1 protein could bind to E2F1-binding BS3 site on Smac promoter and participated in the formation of a proteins-DNA complex. Moreover, in the complex, E2F1, not Par-4, was found to be directly bound to the Smac promoter, suggesting that Par-4 exerted indirectly its transcriptional control on the Smac gene though interacting with E2F1. Expression of full-length Par-4 in human-bone mesenchymal cells resulted in increased activity of the Smac promoter. In addition, the indirect transcripional regulation of Par-4 on Smac depended on its COOH terminus-mediated interaction between Par-4 and E2F1. We conclude that the formation of proteins–DNA complex, containing Par-4 protein, E2F1 protein and the Smac promoter, contributes to the pro-apoptotic effect on glutamate-treated human-bone mesenchymal stem cells.

Effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells

To investigate the effects of Par-4 gene silencing induced by siRNA on the expression of Smac gene, activity of caspase-3, and apoptosis of human bone marrow mesenchymal stem cells (hBMSCs). Bone marrow was obtained from a healthy young man and hBMSCs were isolated and cultured. Two siRNAs (Par-4-siRNA-1 and -2) targeting Par-4 gene were chemically synthesized. Eukaryocytic expression vectors containing these Par-4 siRNA sequences were established and transfected into the hBMSCs. The hBMSCs were divided into 4 groups: non-transfected hBMSCs (normal control group), blank Pae-4 plasmid transfected hBMSCs (Par4 control group), Par4-siRNA-1 transfected hBMSCs, and Par-4-siRNA-2 transfected hBMSCs. The expression of Par-4 mRNA was detected by real-time PCR. Another hBMSCs were inoculated in DMEM and divided into 4 groups: non-transfected normal hBMSCs, glutamate (an apoptosis inducer) + non-transfected hBMSC group, glutamine + Par-4-siRNA-1 hBMSC group, and glutamate + Par4-SiRNS-2 hBMSC group. Flow cytometry was used to detect the apoptotic rate. The relative activity of caspase-3 was determined by colorimetric assay. Western blotting was used to detect the Smac protein expression. The relative mRNA expression levels of Par-4 gene of the Par-4-siRNA-1 hBMSCs, Par-4 SiENA-2 hBMSCs, and Par-4 control hBMSCs were 0.12 +/- 0.03, 0.33 +/- 0.09, and 0.97 +/- 0.02 respectively, decreased by 88%, 67%, and 3% respectively compared with that of the normal control. The percentages of apoptotic cells of the glutamate + Par-4-siRNA-1 hBMSCs was (37.2 +/- 6.3)%, significantly lower than that of the glutamate + non-transfected hBMSC group [(58.9 +/- 8. 9)%, F = 58.26, P < 0.01). The Smac protein expression level of the glutamate + non-transfected hBMSC group was significantly higher than that of the normal control group (P < 0.01); however, the Smac protein expression level of the Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected hBMSC group (P < 0.01). The caspase-3 activity of the glutamate + Par-4-siRNA-1 hBMSC group was significantly lower than that of the glutamate + non-transfected BMSC group (P < 0.01). Par-4-siRNA-1 inhibits markedly the apoptosis of the hBMSCs induced by glutamate. Par-4 gene silencing induced by siRNA inhibits the apoptosis of hBMSCs. The mechanism of the inhibition may be closely related to suppression of the up-regulation of Smac gene expression and caspase-3 activity.

Construction of Smac gene-containing and human prostate specific antigen promoter-regulated vector and its expression

Objective To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen (PSA)enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods PSA enhancer (PSA) and promoter (PSAP) sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion An expression vector containing the Smac gene (based on elements of the PSA gene regulatory sequences) has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.