Effect of hepatitis B virus X on the apoptosis of placental trophoblastic cells and its potential mechanism / 西安交通大学学报(医学版)

Objective To investigate the effect of hepatitis B virus X (HBx)protein on the apoptosis of placental trophoblastic cells and its potential mechanism.Methods A pcDNA3.1 expression vector of HBx gene was constructed and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After transfection for 48 h,RT-PCR and immunofluorescence analyses were made to detect HBx mRNA and protein expressions.Flow cytometry was used to detect the early apoptosis status of JEG-3 and HTR-8 cells.The expressions of PI3K and p-Akt were detected by immunofluorescence and Western blotting.Results After transfection for 48 h,RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expressions were detected in JEG-3 and HTR-8 cells.Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection (P <0.05).Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells (P < 0.05 ).Conclusion HBx gene can be transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After the transfection,the early apoptosis of JEG-3 and HTR-8 cells is reduced.Its inhibition on apoptosis is related to the activation of the PI3K/Akt signaling path-way.

Relationship between expression of CD142 protein and the promoter methylation in the placenta in severe preeclampsia / 西安交通大学学报(医学版)

Objective To explore the relationship between expression of CD142 protein and the promoter methylation in the placenta of severe preeclampsia patients.Methods We assessed 24 patients complicated with severe preeclampsia as case group and 24 normal pregnant women as control group via qRT-PCR, immunohistochemistry and Western blotting for CD142 expression and MSP technology for methylation in CD142 promoter region.Results The relative expression quantity of CD142 mRNA in severe preeclampsia group (1.45± 0.42)was higher than that in normal group (0.25±0.28)(P <0.05).The expression quantity of CD142 protein in severe preeclampsia group (0.857±0.043)was higher than that in normal group (0.248 ±0.035)(P <0.05).The positive rate of CD142 promoter region methylation was lower in severe preeclampsia group than in normal group (29.2% vs .100.0%,χ2 =36.11,P <0.001)while the positive rate of CD142 promoter region unmethylation was higher than that in the latter group (100.0% vs .20.8%,χ2 =29.85,P <0.001).A negative correlation was found between the CD142 promoter methylation level and the expression quantity of CD142 protein (r = -0.909,P <0.05).Conclusion The expression of CD142 protein regulated by the promoter methylation plays a crucial role in the mechanism of severe preeclampsia.

Effect of hepatitis B virus X on the apoptosis of placental trophoblastic cells and its potential mechanism / 西安交通大学学报(医学版)

Objective To investigate the effect of hepatitis B virus X (HBx)protein on the apoptosis of placental trophoblastic cells and its potential mechanism.Methods A pcDNA3.1 expression vector of HBx gene was constructed and transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After transfection for 48 h,RT-PCR and immunofluorescence analyses were made to detect HBx mRNA and protein expressions.Flow cytometry was used to detect the early apoptosis status of JEG-3 and HTR-8 cells.The expressions of PI3K and p-Akt were detected by immunofluorescence and Western blotting.Results After transfection for 48 h,RT-PCR and immunofluorescence analyses showed that HBx mRNA and protein expressions were detected in JEG-3 and HTR-8 cells.Flow cytometry revealed that early apoptosis of JEG-3 and HTR-8 cells was reduced by pcDNA-HBx transfection (P <0.05).Immunofluorescence and Western blotting showed that PI3K and p-Akt were significantly upregulated in HTR-8 cells (P < 0.05 ).Conclusion HBx gene can be transfected into JEG-3 and HTR-8 human placental trophoblastic cell lines,respectively.After the transfection,the early apoptosis of JEG-3 and HTR-8 cells is reduced.Its inhibition on apoptosis is related to the activation of the PI3K/Akt signaling path-way.

Relationship between expression of CD142 protein and the promoter methylation in the placenta in severe preeclampsia / 西安交通大学学报(医学版)

Objective To explore the relationship between expression of CD142 protein and the promoter methylation in the placenta of severe preeclampsia patients.Methods We assessed 24 patients complicated with severe preeclampsia as case group and 24 normal pregnant women as control group via qRT-PCR, immunohistochemistry and Western blotting for CD142 expression and MSP technology for methylation in CD142 promoter region.Results The relative expression quantity of CD142 mRNA in severe preeclampsia group (1.45± 0.42)was higher than that in normal group (0.25±0.28)(P <0.05).The expression quantity of CD142 protein in severe preeclampsia group (0.857±0.043)was higher than that in normal group (0.248 ±0.035)(P <0.05).The positive rate of CD142 promoter region methylation was lower in severe preeclampsia group than in normal group (29.2% vs .100.0%,χ2 =36.11,P <0.001)while the positive rate of CD142 promoter region unmethylation was higher than that in the latter group (100.0% vs .20.8%,χ2 =29.85,P <0.001).A negative correlation was found between the CD142 promoter methylation level and the expression quantity of CD142 protein (r = -0.909,P <0.05).Conclusion The expression of CD142 protein regulated by the promoter methylation plays a crucial role in the mechanism of severe preeclampsia.

Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism.</p><p><b>METHODS</b>Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot.</p><p><b>RESULTS</b>NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner.</p><p><b>CONCLUSION</b>NNAMB induces apoptosis via mitochondrial pathway in K562 cells.</p>

Immunotherapy of non-Hodgkin's lymphomas (NHL) by anti-CD22 antibody--review / 中国实验血液学杂志

CD22 is a transmembrane sialoglycoprotein and a member of the immunoglobulin superfamily. Its expression is restricted to the B cell lineage and a vast majority of B cell NHLs. CD22 plays a key role in B cell development, survival, and function. Humanized anti-CD22 antibodies were developed to minimize the immunogenicity and to enhance effector interactions during their developments of diagnostic and immunotherapeutic agent. Preclinical test with anti-CD22 antibodies indicates that a single, conjugated or radiolabeled agent has shown preliminary antitumor activity in patients with recurrent and heavily pretreated NHL. Anti-CD22 antibodies were well tolerated, without dose-dependant toxicity. Anti-CD22 antibodies are currently being evaluated in combination with rituximab, and the early results suggest that the combination of the two antibodies are well tolerated and may result in better clinical activity than the single agent alone. Thus, anti-CD22 antibodies are theoretically good candidates alone and in combination with other drugs in the treatment of B cell malignancies. In this review, the physiologic function and characteristics of CD22 antigen as target molecule of guide therapy for NHL, the types of anti-CD22 antibodies in therapy of NHL and the combination use with other antibodies were summarized.

Primary study on the anti-tumor effect of ethanol extracts of Solanum lyratum / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the anti-tumor activity of the ethanol extracts of Solanun lyratum in vitro and in vivo.</p><p><b>METHOD</b>In vitro, the inhibitory effects of ethanol extracts of S. lyratum on proliferation of human hepatoma BEL-7402 cell and gastric carcinoma SGC-7901 cell were measured by MTT colorimetric assay. The mouse tumor model was used to investigate the effects of ethanol extracts on tumor growth.</p><p><b>RESULT</b>The studies demonstrated that ethanol extracts of S. lyratum inhibited proliferation of BEL-7402 cells and SGC-7901 cells, and the IC50 values on them were (287.40 +/- 5.84) micron x mL(-1) and (176.14 +/- 5.18) microg x mL(-1), respectively. The tumor inhibitory rate of high doses of ethanol extracts on S180 sarcoma-transplanted mice and H22 hepatic cancer were (41.15 +/- 4.54) % and (45.00 +/- 7.37) %, respectively. When the dose of ethanol extracts varied from low to high, it was able to inhibit the growth of S180 sarcoma-transplanted mice and H22 hepatic cancer in a dose-dependent manner.</p><p><b>CONCLUSION</b>In tumor inhibitory test, it was shown that the ethanol extracts of S. lyratum may possess significantly inhibitory effect in vitro and in vivo. No acute toxic effect was found in our experiment.</p>

IL-1beta Acts in Synergy with Endogenous IL-1beta in A375-S2 Human Melanoma Cell Apoptosis Through Mitochondrial Pathway

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.

Pseudolaric acid B induces human melanoma A375-S2 cell apoptosis in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To study the mechanisms of pseudolaric acid B-induced apoptosis on A375-S2 cells.</p><p><b>METHOD</b>MTT, fluorescence microscope observation, DNA agarose gel electrophoresis and Western blot analysis wereused.</p><p><b>RESULT</b>Pseudolaric acid Binduces A375-S2 cell apoptosis in a time and dose-dependent manner. Apoptotic bodies and DNA ladder were observed in 5 micromol x L(-1) pseudolaric acid B-treated A375-S2 cells for 36 h. The expression of Bcl-2, Bcl-xL and ICAD was reduced time dependently, whereas the expression of Bax was increased.</p><p><b>CONCLUSION</b>The major cause of pseudolaric acid B induced cytotoxicity on A375-S2 cells was apoptosis. Mitochondria proteins and ICAD might be involved in the apoptotic pathways of pseudolaric acid B-treated A375-S2 cells.</p>

Effect of salidroside on mitochondria injury induced by sodium azide / 药学学报

<p><b>AIM</b>To study the protective effect of salidroside on mitochondria injury induced by sodium azide.</p><p><b>METHODS</b>Human neuroblastoma SH-SY5Y cells were exposed to sodium azide with different concentration of salidroside, then cell viability was measured by thiazolyl blue (MTT) method and mitochondrial membrane potential (MMP) was detected by JC-1 method. Protective effect of salidroside against disfunction of mitochondria induced by sodium azide was detected by resazurin method.</p><p><b>RESULTS</b>After exposing to 64 mmol x L(-1) sodium azide for 4 hours, cell viability and MMP of SH-SY5Y cells significantly decreased. When pretreated with salidroside, the cell damage was greatly reduced and the mitochondrial membrane potential was maintained. Furthermore, salidroside can protect function of rat brain mitochondria against damage induced by sodium azide.</p><p><b>CONCLUSION</b>Salidroside was demonstrated to play an important role in improving the function of mitochondria.</p>

Mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells.</p><p><b>METHODS</b>MTT assay, fluorescence microscopy, DNA agarose gel electrophoresis, flow cytometry and Western blot analysis were carried out to assess the morphological and biochemical changes of A375-S2 cells after capsaicin treatment.</p><p><b>RESULTS</b>Capsaicin induced A375-S2 cell death in a time- and dose-dependent manner. Sub-diploid peak was seen at 24 h after 250 micromol/L capsaicin treatment, and apoptotic bodies and DNA ladder were observed at 36 h after capsaicin treatment. The expression of inhibitor of caspase activated DNase (ICAD) was reduced with the lapse of time.</p><p><b>CONCLUSION</b>Capsaicin induces A375-S2 cell apoptosis and down-regulation of ICAD contributes to this process.</p>

Apoptosis-inducing effect of carbazole alkaloid (HY-1) in human erythroleukemia K562 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate apoptosis-inducing effect and its mechanisms of HY-1, a carbazole alkaloid, on human erythroleukemia K562 cells.</p><p><b>METHODS</b>Cell proliferation was detected by sulforhodamine B (SRB) assay after treated with HY-1 at indicated doses. Cell cycle analysis was performed by flow cytometry, mitochondria membrane voltage change was assessed by rhodamine 123 staining, annexin V-PI apoptosis detecting kit and DNA agarose gel electrophoresis were used to identify apoptosis-inducing effect of HY-1. The alterations of apoptosis-relating proteins were detected by Western blot.</p><p><b>RESULTS</b>The IC(50) of HY-1 in K562 cells was (29.05 +/- 0.90) micromol/L by SRB assay. HY-1 had significant apoptotic inducing effect on K562 cells in a dose- and time-dependent manner as verified by appearance of Sub-G(1) peak on histogram of flow cytometry analysis, reduction of mitochondria membrane voltage, appearance of double positive cell group in Annexin V-PI apoptosis detecting test, and remarkable DNA ladder. The expression of cytosolic cytochrome c was apparently increased. Pro-caspase-9, pro-caspase-3 and PARP were all cleaved to active segments. There was no change in the expression of caspase-8.</p><p><b>CONCLUSION</b>HY-1 exerts its anticancer activity through triggering apoptosis of K562 cells by mitochondria-activating pathways.</p>

Effect of protein kinase C on human melanoma A375-S2 cell death induced by evodiamine / 药学学报

<p><b>AIM</b>To study the role of PKC in evodiamine-induced A375-S2 cell death.</p><p><b>METHODS</b>Ratio of apoptosis induced by evodiamine was determined by TUNEL assay. MTT assay was carried out to assess cytotoxic effect of evodiamine. The influence on expression of ERK, phospho-ERK and Bcl-2 was detected by Western blotting analysis.</p><p><b>RESULTS</b>TUNEL assay indicated that apoptosis was the type of A375-S2 cell death induced by evodiamine treatment for 24 h. Both staurosporine (inhibitor of PKC) and PD98059 (inhibitor of ERK) cooperated with evodiamine to further induce A375-S2 cell death. Evodiamine inhibited PKC activity, down-regulated the expression of ERK, phospho-ERK and Bcl-2, and staurosporine was capable of augmenting these effects induced by evodiamine.</p><p><b>CONCLUSION</b>PKC lies upstream and exhibits regulatory effect on ERK and Bcl-2 in evodiamine-induced cell death.</p>

Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We have reported that norcantharidin (NCTD) induces human melanoma A375-S2 cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in the apoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD.</p><p><b>METHODS</b>We assessed the effects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay, DNA fragmentation (DNA agarose gel electrophoresis), and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were also collected.</p><p><b>RESULTS</b>The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKC inhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment with NCTD, and inhibitors of c-Jun NH2-terminal kinase (JNK) and p38 (SP600125 and SB203580, respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38 expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation of phosphorylated JNK and phosphorylated p38, but had little effect on extracellular signal-regulated kinase (ERK) expression.</p><p><b>CONCLUSION</b>These results suggest that the activation of JNK and p38 MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays an important regulation role in the activation of MAPKs.</p>

Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.

Mechanism of dracorhodin perchlorate-induced Hela cell apoptosis / 药学学报

<p><b>AIM</b>To study the mechanism of dracorhodin perchlorate-induced Hela cell apoptosis.</p><p><b>METHODS</b>Cell viability was measured by MTT method. Morphological changes were observed by phase contrast microscopy and Hoechst 33258 staining. DNA fragmentation was assayed by agarose gel electrophoresis. Protein expression was detected by Western blot analysis.</p><p><b>RESULTS</b>Dracorhodin perchlorate induced Hela cell apoptosis. The apoptosis was partially reversed by caspase-1, -3, -8, -9 and caspase family inhibitors. Treatment of Hela cells with dracorhodin perchlorate for 12 h increased the protein expression ratio of Bax/Bcl-XL; procaspase-3, -8, ICAD and PARP were cleaved to smaller molecules.</p><p><b>CONCLUSION</b>Dracorhodin perchlorate induced Hela cell death via alteration of Bax/Bcl-XL ratio and activation of caspases.</p>

Anti-emetic effect of ethanol extract from "Wuzhuyu broth" / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the anti-emetic effect of ethanol extract from "WuZhuYu broth" and its mechanism.</p><p><b>METHOD</b>Three experiments were carried out test which extract has anti-emetic activity, such as CuSO4-induced pigeon's emetic response, gastric emptying in mice and ACh-induced or 5-HT-induced contraction in vitro gastric muscle in rats. Meanwhile, effect of anti-emetic extract on concentration-response curve to ACh, 5-HT, histamine was investigated.</p><p><b>RESULT</b>50% ethanol extract and 70% ethanol extract were identified as having significantly stronger anti-emetic activities with little side effect, which showed the significant effect on concentration-response curve to ACh, 5-HT, histamine.</p><p><b>CONCLUSION</b>50% ethanol extract and 70% ethanol extract contain more anti-emetic fractions, more anti-emetic fractions can be gained at the concentrations of 50% and 70% ethanol; the mechanism of anti-emetic effect is related to its antagonism to the receptors of ACh, 5-HT, histamine.</p>