ZM-66, a New Podophyllotoxin Derivative Inhibits Proliferation and Induces Apoptosis in K562/ADM Cells

Objective To investigate the anti-tumor effect of ZM-66 on multidrug-resistant leukemic cell line K562/ADM. Methods The K562/ADM cells were treated with varying concentrations （0, 1, 2, 4×10^-3 mmol/L） of ZM-66 or etoposide for 24 hours. The proliferation was detected by Sulforhodamine B Sodium Salt （SRB） assay and apoptosis was detected by flow cytometry analysis and fluorescent staining. In addition, the expression levels of p53 and bax genes in K562/ADM cells were detected by RT-PCR analysis. The level of P-glycoprotein （P-gp）, P53 and Bax protein in K562/ADM cells were detected by Western blot assay. Results SRB assay demonstrated that etoposide had little inhibitory effect on K562/ADM cells, whereas ZM-66 （1, 2, 4×10^-3 mmol/L） had significantly inhibitory effect on K562/ADM cells （all P〈0.01）. The acridine orange/propidium iodide dual staining showed that there were typical condensation of chromatin and nuclear fragmentation nuclei with red color in ZM-66 treated cells. Flow cytometric analysis showed that there was a significantly increase of apoptotic cells i~ K562/KDM cells after treated with ZM-66. RT-PCR showed that the p53 and bax mRNA expression levels in K562/ADM cells treated with ZM-66 at 1, 2, 4×10^-3 mmol/L were higher than those in the cell without treatment. Western blot showed that the P53 and Bax protein expression levels in K562/ADM cells treated with ZM-66 at 2, 4x 10 s mmol/L were higher than those in the cell without treatment. But the P-gp protein expression level in K562/ADM cells treated with ZM-66 at 2, 4×10^-3 mmol/L was gradually lower than those in the cell without treatment. Conclusion ZM-66 is able to induce cell death by apoptosis in vitro, as a result of the reverse of theapoptosis resistance in drug-resistant K562/ADM cells by modulating expression of key factors associated with apoptosis induction.     

The Apoptosis of Bovine Lens Epithelial Cells Induced by Proteasome Inhibitor MG132

To investigate the effect of proteasome inhibitor MG132 on the apoptosis of bovine lens epithelial cells （BLECs）, the cells were treated with MG132 at different concentrations for12, 24 and 36 h. The cell viability was analyzed by MTT assay and the effect of MG132 on the apoptosis of BLECs was assessed by flow cytometry （FCM）. The results showed that after treatment for the same period, the inhibitory effect of MG132 on BLECs proliferation was enhanced with the increment of the concentration of MG132 （0, 2, 5, 10, μmol/L） （P〈0.05）. The 50% inhibiting concentration （IC50） was 2.03 μmol/L when the BLECs were treated with MG132 for 36 h. MG132 also induced the apop- tosis of BLECs obviously. FCM showed that the apoptosis index of the cells treated by MG132 at 2 μmol/L for 12 h was （20.24±1.51）%, and that of the control was （0.98±0.20）% respectively （P〈0.01, n=3）. It was concluded that MG132 could lead to apoptosis of BLECs. The decrease of proteasome activity may play an important role in the formation and development of cataract.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor(NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated.The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed.The in vitro cultured A549 cells were transfected with pcDNA3.1( )/IκBα alone,or pcDNA3.1( )/IκBα combined with cisplatin.The mito-chondrial membrane potential(?ψm) was determined by rhodamine 123,the activity of caspase-3 was tested by colorimetric assay,and cell apoptosis was detected by flow cytometry with the annexin Ⅴ/propidium iodide assay.The results showed that the activity of NF-κΒ in A549 cells was inhibited by transfecting pcDNA3.1( )/IκΒα.Transfection of pcDNA3.1( )/IκΒα alone did not promote apoptosis.Treatment of cisplatin alone had a little effect on cell apoptosis.Transfection of pcDNA3.1( )/IκΒα combined with cisplatin treatment significantly induced apoptosis of A549 cells.It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Altered gene expression reveals molecular mechanisms underlying oridonin-induced apoptosis of multiple myeloma LP-1 cells

Objective: To investigate the effect of oridonin on proliferation and invasion of human multiple myeloma LP-1 cells and the underlying mechanism. Methods: LP-1 cells in culture medium in vitro were treated with oridonin at the different concentration. Cell proliferation was measured by Microwave Theory and Techniques (MTT) assay and cell apoptotic rate was detected by flow cytometry. Morphology of cell apoptosis was observed by transmission electron microscope. Expressions of Bax, Bcl-2, Caspase-3, NF-κB as well as I-κB mRNA were detected by real-time PCR. Results: The MTT assays and flow cytometry revealed that oridonin could inhibit the growth of LP-1 cells and cause apoptosis significantly; the suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis were found under a transmission electron microscope after the cells were treated with oridonin at 25 μmol/L for 24 h. Along with the apoptotic process, Bcl-2, Caspase-3,NF-κB gene expressions were down-regulated (P<0.05). On the contrast, the Bax and I-κB gene expressions were up-regulated (P<0.05). Conclusion: Oridonin could inhibit the proliferation of LP-1 cells via inducing apoptosis. We concluded that oridonin induces apoptosis in LP-1 cells via activation of caspase-3 as well as down-regulation of Bcl-2 and up-regulation of Bax expression. The results suggested that oridonin could induce apoptosis of LP-1 cells through mitochondria- and caspase3-dependent pathways. Meanwhile, the inhibition of NF-κB and the activation of I-κB indicate pro-apoptotic stimuli. In one word, oridonin might be an important potential anti-myeloma reagent.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were trans-fected with pcDNA3.1( )/IκBα alone, or pcDNA3.1( )/IκBα combined with cisplatin. The mitochondrial membrane potential (△ψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1( )/IκBα. Transfection of pcDNA3.1( )/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1( )/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Inhibitory Effects of Parthenolide on the Activity of NF-κB in Multiple Myeloma via Targeting TRAF6

This study examined the mechanism of the inhibitory effect of parthenolide(PTL) on the activity of NF-κB in multiple myeloma(MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6(TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.     

Anticancer Activities of Trichostatin A on Maligant Lymphoid Cells

The anticancer activity of trichostain A (TSA) on human B cell non-Hodgkin's lymphoma and its mechanism were explored. The effect of TSA on the growth of Raji cells and normal peripheral blood mononuclear cells (NPBMNC) was studied by MTT assay. The effect of TSA on the apoptosis of Raji cells and NPBMNC was studied by flow cytometry and TDT-mediated dUTP nick end labeling (TUNEL). The effect of TSA on the cell cycle of Raji cells was studied by propidium iodide method. The results showed that TSA potently inhibited proliferation of Raji cells at microgram concentrations and induced apoptosis of Raji cells in a time- and concentration-dependent manner. Treatment with TSA induced accumulation of cells in G0/G1 or G2/M and a concomitant decrease of cell population in S phase. However, NPBMNC was less sensitive to the cytotoxic effect of TSA than Raji cells. It was concluded that TSA may inhibit the proliferation of Raji cells by regulating the cell cycle and inducing the cell apoptosis. Moreover, TSA demonstrates low toxicity in NPBMNC but selectively induces apoptosis of Raji cells.     

Anisodamine Inhibits Engotoxin—induced Tissue Factor Expression in Human Endothelial Cells

By study on the effect of anisodamine on lipopolysaccharide-induced expression of tissue factor(TF) in vascular endothelial cells (EC),the mechanism of anisodamine antithrombosis,as well as in the treatment of bacteraemic shock was investigated.Human umbilical vein endothelial cells (HUVECs) were cultured by trypsin digestion method.TF activity was measured in the lysates of HUVEC by using a single step clotting assay.Specific mRNA expression was detected by Northern blotting.In order to evaluate a possible contribution of the nuclear factor (NF)-κB pathway on the effects observed,electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVECs and NF-κB-binding oligonucleotides.The results showed that treatment of HUVEC with LPS resulted in a significant increase in TF activity.Anisodamine dose-dependently inhibited LPS-induced upregulation of TF.These effects was also confirmed on the level of specific TF mRNA expression by Northern blotting.Furthermore,EMSA showed that anisodamine completely abolished LPS-induced NF-κB DNA binding activity in nuclear extracts from HUVECs treated with LPS together with anisodamine.The results suggest that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced TF expression in these cells.Its interference with the NF-κB pathway might-at least in part-contribute to this effect.The ability of anisodamine to counteract LPS effect on endothelial cells might be one underlying mechanism explaining its antithrombosis and efficacy in the treatment of bacteraemic shock.     

Effect of DRB on the Biological Characteristics of Human Laryngeal Carcinoma Hep-2 Cell Line

In order to study the effect of 5, 6-Dichloro-1-β-D-ribofuranosyl-benzimidazole (DRB) on the biological characteristics of human laryngeal carcinoma Hep-2 cell line in vitro, Hep-2 cells cultured in vitro were treated with different concentrations of DRB. Changes in cell proliferation, apoptotic rate and invasiveness were detected by MTT assay, flow cytometry (FCM) and matrigel in vitro invasion assay, respectively. It was found that DRB inhibited the proliferation of Hep-2 cells in a dose- and time-dependent manner. After being treated with 0, 10, 20, 40, 80 μmmol/L DRB for 24 h, the apoptotic rate in Hep-2 cells was (0.68±0.19)%, (1.95±0.12)%, (8.51±0.26)%, (11.26±0.17)% and (14.99±0.32)%, respectively. The matrigel in vitro invasion assay revealed that DRB began to inhibit the invasion of Hep-2 cells at the concentration of 5 μmmol/L, and with the increase of DRB concen-tration, the inhibitory effect was enhanced. It was suggested that DRB could influence the essential biological characteristics of Hep-2 cells, inhibit Hep-2 cells proliferation, reduce invasive ability and induce apoptosis of Hep-2 cells.     

Effect of Retinoic Acid on Apoptosis and Expression of Fas Proteins in Mouse Blastocysts Cultured In Vitro

Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid （RA） for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL-FITC） assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm （TE） and inner cell mass （ICM） cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA （10 μmol/L） resulted in a more significant apoptosis and higher expression level of Fas proteins （P〈0.01）. It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.     

Holotransferrin enhances selective anticancer activity of artemisinin against human hepatocellular carcinoma cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.     

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha -induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65,IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB.TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.     

Inhibitory activity of nuclear factor-κB potentiates cisplatin-induced apoptosis in A549 cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti-proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow eytometry. The cell cycle of the tumor cells was also observed by flow eytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatoeareinoma cells. Incubated with melatonin, ehromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G0/S increased but that of G0/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatoearcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.     

Regulating effects of arsenic trioxide on cell death pathways and inflammatory reactions of pancreatic acinar cells in rats

Background It is accepted that inflammatory cytokines play a key role in the development of acute pancreatitis, so blocking the initiation of inflammatory reactions may alleviate pathological changes of acute pancreatitis. We studied the regulatory effect of arsenic trioxide （As2O3） on apoptosis and oncosis of pancreatic acinar cells in vitro and in vivo and its therapeutic effect on acute pancreatitis. Methods Pancreatic acinar cells were isolated by collagenase digestion method. Apoptosis and oncosis of isolated pancreatic acinar cells were detected with Hoechst 33258＋PI or Annexin V＋PI double fluorescent staining. Amylase and lactate dehydrogenase release were measured. Acute pancreatitis was induced in Wistar rats by intraperitoneal injections of caerulein, and apoptosis was detected with terminal dUTP nick-end labeling method. Tumor necorsis factor α （TNF-α） mRNA, myeloperoxidase, nuclear factor-κB and histological grading of pancreatic damage were measured. Results There was an increased apoptosis but a decreased oncosis of pancreatic acinar cell after the treatment with AS2O3. The levels of lactate dehydrogenase and amylase release were markedly decreased in As2O3 treated group. Myeloperoxidase content, TNF-α mRNA level, nuclear factor-κB activation and pathological score in As2O3 treated group were significantly lower than in the untreated group. Conclusions As2O3 can induce apoptosis and reduce oncosis of pancreatic acinar cell, thus resulting in reduced release of endocellular enzyme of acinar cells, reduced inflammatory cell infiltration and decreased the production of inflammatory cytokines, so that the outcome of alleviated pathological changes was finally achieved.     

Triptolide-induced apoptosis by inactivating nuclear factor-kappa B apoptotic pathway in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

The effect of triptolide on proliferation and apoptosis of human multiple myeloma RPMI-8226 cells in vitro,as well as the roles of nuclear factor-kappa B(NF-κB) and IκBα was investigated.The effect of tritptolide on the growth of RPMI-8226 cells was studied by MTT assay.Apoptosis was detected by Hoechest 33258 staining and Annexin V/PI double staining assay.The expression of NF-κB and IκBα was observed by Western blot and confocal microscopy.The results showed that triptolide inactivated NF-κB apoptotic pathway in human multiple myeloma RPMI-8226 cells.Triptolide at nM range induced proliferation inhibition in a dose-and time-dependent manner and apoptosis in a dose-dependent fashion in RPMI-8226 cells.Besides,we observed the inhibition of NF-κB /p65 in the nuclear fraction was correlated with the increase in the protein expression of IκBα in the cytosol.These results suggested that triptolide might exhibit its strong anti-tumor effects via inactivation of NF-κB/p65 and IκBα.