Apoptosis Regulatory Proteins ; Apoptosis ; Antiviral Agents/pharmacology* ; Mitochondrial Proteins ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology*

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

OBJECTIVE: To observe the effect of programmed cell death 5 (PDCD5) protein on the apoptosis of multiple myeloma KM3 cells induced by dexamethasone and to understand its mechanism. METHODS: The human recombinant PDCD5 (rhPDCD5) protein was added (alone of different concentrations or associated with dexamethasone) into KM3 cells. Cultured together for certain time, the cells were collected for the following experiments: (1)The effect of rhPDCD5 protein and dexamethasone on the apoptotic rate of KM3 cells was determined by flowcytometry (FCM) analysis after the cells were stained by Annexin V-FITC & PI (propidium iodide). (2)Caspase-3 activity of KM3 cells was evaluated by Western blot. (3)The expression of survivin protein in KM3 cells was detected by immunocytochemistry. RESULTS: The apoptotic rate of KM3 cells and the activity of caspase-3 increased significantly, and that treated with rhPDCD5 protein and dexamethasone was higher than that treated with rhPDCD5 protein only. The expression of survivin protein in the rhPDCD5 with dexemethas group was down-regulated, and with the concentration of rhPDCD5 and dexamethasone increasing, the changes was more obviously. CONCLUSION PDCD5 protein can induce the apoptosis of KM3 cells, and accelerate the apoptosis of KM3 cells induced by dexamethasone. PDCD5 protein may reduce the expression of survivin protein and increase activation of caspase-3 to play its role in promoting apoptosis.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Drug Synergism ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Multiple Myeloma ; pathology ; Neoplasm Proteins ; pharmacology ; Recombinant Proteins ; pharmacology ; Survivin

The study was aimed to investigate the effect of artesunate (ART) on the apoptosis of HL-60 cells in vitro and the expression of Bcl-2 and ICAD in the process of apoptosis induced by ART. The inhibition of ART on HL-60 cells were evaluated by means of MTT assay; cell apoptosis was detected by light microscopy, agarose gel electro-phoresis, flow cytometry; Western blot was used to analyze the expression of Bcl-2 and ICAD in cells during apoptosis induced by ART. The results showed that ART could significantly inhibit the proliferation of HL-60 cells in time-and dose-dependent manner. After treating HL-60 cells with ART for 48 hours, the IC(50) values was 18.33 microg/ml and its inhibition effect contributed to the induced apoptosis. Bcl-2 and ICAD proteins both all expressed in HL-60 cells, the level of expression declined as concentration increased. It is concluded that artesunate may induce apoptosis of HL-60 cells in vitro, Bcl-2 and ICAD may be an important control factor in the signal transduction pathway of ART-induced apoptosis.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Artemisinins ; pharmacology ; HL-60 Cells ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism

The expression levels of programmed cell death 5 (PDCD5) are down-regulated in many malignancies. SG611-pdcd5, a recombinant conditionally replicative adenovirus carrying pdcd5 gene expression cassette, can evidently kill the leukemic cells and protect selectively the normal cells. The purpose of this study was to investigate the synergistic killing effect of SG611-pdcd5 and low-dose etoposide (VP-16) on K562 cells. K562 cells were treated with different concentrations of VP-16 or different multiplicities of infection (MOI) of SG611-pdcd5. After 48 hours of incubation the cell viability was determined by using MTT assay. The results showed that the cell viability of SG611-pdcd5 (MOI = 40) plus VP-16 (0.5 µg/ml) group significantly decreased as compared with single SG611-pdcd5 (MOI = 40) treatment group or single VP-16(0.5 µg/ml) treatment group (42.00 ± 5.75% vs 59.45 ± 4.12%; 42.00 ± 5.75% vs 82.91 ± 3.41%, respectively, both p < 0.05). The synergistic killing effect of SG611-pdcd5 plus VP-16 was higher than that of PDCD5 protein plus VP-16 or that of non-replicating adenovirus carrying pdcd5 (Ad-pdcd5) plus VP-16 (both p < 0.05). The cell viability of VP-16 (4.0 µg/ml) plus SG611-pdcd5 (MOI = 40) group, VP-16 (4.0 µg/ml) plus proPDCD5 (40 µg/ml) group and VP-16 (4.0 µg/ml) plus Ad-pdcd5 (MOI = 80) group was 37.09 ± 1.89%, 52.36 ± 1.64% and 73.64 ± 4.33%, respectively. It is concluded that SG611-pdcd5 can promote K562 cell death induced by low-dose VP-16. The combination of SG611-pdcd5 and VP-16 can enhance the killing effect on leukemic cells.
Adenoviridae ; genetics ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; Cell Survival ; Etoposide ; pharmacology ; Genetic Vectors ; Humans ; K562 Cells ; Neoplasm Proteins ; genetics

To explore the mechanism of autophagic death of acute myelocytic leukemia cell U937 induced by clofarabine, the MTT bioassay was used to analyze the growth inhibitory effect and half inhibition concentration on U937 incubated in vitro with different concentrations of clofarabine for 24 and 48 hours, and the flow cytometry was used to detect the autophagy rate of U937. The expression of Beclin 1 in U937 treated by clofarabine for 48h was measured by Western blot. The results indicated that when U937 cells were treated with 0.01 µmol/L and 0.15 µmol/L clofarabine for 48 hours, the proliferation inhibition rate was 46.92% ± 4.24% and 86.10% ± 1.16%, and the half inhibition concentration of clofarabine was 0.022 µmol/L. With 0.01 µmol/L and 0.1 µmol/L clofarabine on U937 for 48 hours, the autophagy rate was 11.0033% ± 1.4387% and 59.4133% ± 3.5409%, and increased in dose-dependent manner (r = 0.99). Meanwhile the Beclin 1 was upregulated along with increase of clofarabine concentration, as compared with control group, the difference was statistically significant (P < 0.05). It is concluded that the different concentrations of clofarabine can significantly inhibit the proliferation of U937 in dose-dependent manner, and the mechanism of autophagic cell death in U937 may be associated with the upregulation of Beclin 1 expression.
Adenine Nucleotides ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Arabinonucleosides ; pharmacology ; Autophagy ; drug effects ; Beclin-1 ; Cell Proliferation ; drug effects ; Humans ; Membrane Proteins ; metabolism ; U937 Cells

OBJECTIVETo examinie the synergistic effects of Banxia Xiexin Decoction (, Known as Banhasasim-tang in Korean) extract (BXDE) on cisplatin-induced cytotoxicity in the A549 human lung cancer cell lines.

METHODSA549 cells were treated with varying concentrations (50-200 μg/mL) of cisplatin and BXDE alone or in combination for 96 h. We used 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay and flow cytometry to analyze cell viability and apoptosis, respectively.

RESULTSThe exposure of cells to cisplatin and BXDE alone or in combination decreased cell viability dose- and time-dependently (P<0.05), which was found to be mediated by the apoptotic pathway as confirmed by the increase in the annexin V/propidium iodide- stained cell population and a ladder pattern of discontinuous DNA fragments. Furthermore, the apoptosis was inhibited by the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-FMK).

CONCLUSIONSBXDE significantly potentiated apoptotic effects of cisplatin in A549 cells. Moreover, apoptosis induced by BXDE might be the pivotal mechanism mediating its chemopreventative action against cancer.

A549 Cells ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Caspase Inhibitors ; pharmacology ; Cisplatin ; pharmacology ; DNA Fragmentation ; drug effects ; Humans ; Plant Extracts ; pharmacology

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

Sympathetic cues via the adrenergic signaling critically regulate bone homeostasis and contribute to neurostress-induced bone loss, but the mechanisms and therapeutics remain incompletely elucidated. Here, we reveal an osteoclastogenesis-centered functionally important osteopenic pathogenesis under sympatho-adrenergic activation with characterized microRNA response and efficient therapeutics. We discovered that osteoclastic miR-21 was tightly regulated by sympatho-adrenergic cues downstream the β2-adrenergic receptor (β₂AR) signaling, critically modulated osteoclastogenesis in vivo by inhibiting programmed cell death 4 (Pdcd4), and mediated detrimental effects of both isoproterenol (ISO) and chronic variable stress (CVS) on bone. Intriguingly, without affecting osteoblastic bone formation, bone protection against ISO and CVS was sufficiently achieved by a (D-Asp₈)-lipid nanoparticle-mediated targeted inhibition of osteoclastic miR-21 or by clinically relevant drugs to suppress osteoclastogenesis. Collectively, these results unravel a previously underdetermined molecular and functional paradigm that osteoclastogenesis crucially contributes to sympatho-adrenergic regulation of bone and establish multiple targeted therapeutic strategies to counteract osteopenias under stresses.
Adrenergic Agents/pharmacology* ; Apoptosis Regulatory Proteins/pharmacology* ; Bone Diseases, Metabolic/metabolism* ; Humans ; Liposomes ; MicroRNAs/genetics* ; Nanoparticles ; Osteoclasts ; Osteogenesis/physiology* ; RNA-Binding Proteins/pharmacology*

OBJECTIVESurvivin highly overexpresses in the most of human tumors, and it may play an important role in the development of tumor. The aim of this study was to explore the effects of survivin antisense oligonucleotide (ASODN) on the proliferation and the apoptosis of human Hep-2 cell.

METHODSHep-2 cells were transfected with survivin ASODN mediated by lipofectamine, MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide] method was used to observe the cell growth inhibitory rate, the expressions of survivin mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively. Flow cytometry was used to examine cell apoptosis rate. Kinase activity test was used to detect the changes of caspase-3 activity.

RESULTSSurvivin ASODN obviously inhibited the cell growth of Hep-2 cells after transfection. After transfected with survivin ASODN the expressions of survivin mRNA and protein of Hep-2 cells were down-regulated, and apoptosis rate was significantly increased. The activity of caspase-3 increased highly in Hep-2 cells transfected with survivin ASODN, which showed time-dependent.

CONCLUSIONSSurvivin ASODN could inhibit the proliferation of Hep-2 cell and induced apoptosis through down-regulating the the expressions of survivin mRNA and protein.

Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; genetics