OBJECTIVE: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. METHODS: The cytotoxic effects of 28 Nilotinib derivatives on K562, KA, KG, HA and 32D cell lines were detected by MTT assays, and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP RESULTS: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives, which impairs leukemia cell lines, but spares normal hematopoietic cell line. Comparing with Nilotinib, Nilo 22 could induce the apoptosis of GFP CONCLUSION Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells, especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.
Animals ; Apoptosis/drug effects* ; Cell Cycle/drug effects* ; Cell Line, Tumor ; Humans ; Leukemia ; Mice ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Telomerase/metabolism* ; Telomere/metabolism*

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factor-kappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Bortezomib ; pharmacology ; therapeutic use ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; drug effects ; Drug Screening Assays, Antitumor ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Structure-Activity Relationship ; Ubiquitin-Protein Ligases ; genetics ; metabolism

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.
Animals ; Antineoplastic Agents ; isolation & purification ; pharmacology ; toxicity ; Apoptosis ; drug effects ; Caspases ; genetics ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dioscoreaceae ; chemistry ; Heterografts ; drug effects ; growth & development ; Humans ; Inhibitory Concentration 50 ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Nude ; Phosphorylation ; drug effects ; Plant Tubers ; chemistry ; Poly (ADP-Ribose) Polymerase-1 ; metabolism ; Saponins ; isolation & purification ; pharmacology ; toxicity

OBJECTIVETo explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.

METHODSThe HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.

RESULTSThe bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.

CONCLUSIONSBufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/Gphase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.

Apoptosis ; drug effects ; genetics ; Bufanolides ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; drug effects ; genetics ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia, Erythroblastic, Acute ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Up-Regulation ; drug effects ; genetics ; WT1 Proteins ; genetics ; metabolism

Objective: To explore the effect of the AXL expression on the chemosensitivity of prostate cancer PC-3 and DU145 cells to docetaxel and possible mechanisms. METHODS: Using Western blot, we examined the expressions of the AXL protein, p-AXL and Gas6 in the docetaxel-resistant PC-3 (PC-3-DR) and DU145 (DU145-DR) cells stimulated with gradually increased concentrations of docetaxel. We transfected the PC-3 and DU145 cells with negative NC ShRNA and AXL-ShRNA, respectively, which were confirmed to be effective, detected the proliferation, apoptosis and cycle distribution of the cells by CCK8, MTT and flow cytometry after treated with the AXL-inhibitor MP470 and/or docetaxel, and determined the expression of the ABCB1 protein in the PC-3-DR and DU145-DR cells after intervention with the AXL-inhibitor R428 and/or docetaxel. RESULTS: The expression of the AXL protein in the PC-3 and DU145 cells was significantly increased after docetaxel treatment (P <0.05). The expressions AXL and p-AXL were remarkably higher (P <0.05) while that of Gas6 markedly lower (P <0.05) in the PC-3 and DU145 than in the PC-3-DR and DU145-DR cells. The inhibitory effect of docetaxel on the proliferation and its enhancing effect on the apoptosis of the PC-3 and DU145 cells were significantly decreased at 48 hours after AXL transfection (P <0.05). MP470 obviously suppressed the growth and promoted the apoptosis of the PC-3-DR and DU145-DR cells, with a higher percentage of the cells in the G2/M phase when combined with docetaxel than used alone (P <0.05). R428 markedly reduced the expression of ABCB1 in the PC-3-DR and DU145-DR cells, even more significantly in combination with docetaxel than used alone (P <0.05). CONCLUSIONS The elevated expression of AXL enhances the docetaxel-resistance of PC-3 and DU145 prostate cancer cells and AXL intervention improves their chemosensitivity to docetaxel, which may be associated with the increased cell apoptosis in the G2/M phase and decreased expression of ABCB1.
ATP Binding Cassette Transporter, Subfamily B, Member 1 ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Count ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Docetaxel ; Drug Resistance, Neoplasm ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Male ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins ; drug effects ; genetics ; metabolism ; Pyrimidines ; pharmacology ; RNA, Small Interfering ; Receptor Protein-Tyrosine Kinases ; drug effects ; genetics ; metabolism ; Taxoids ; pharmacology

To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells.RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation.Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (<0.05). The cell proliferation was inhibited (<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(=0.478,<0.01).Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.
Adjuvants, Pharmaceutic ; pharmacology ; Carcinoma, Squamous Cell ; drug therapy ; physiopathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Cyclin D1 ; drug effects ; genetics ; DNA-Binding Proteins ; administration & dosage ; pharmacology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; Drug Screening Assays, Antitumor ; methods ; Drug Synergism ; Esophageal Neoplasms ; drug therapy ; physiopathology ; Fluorouracil ; pharmacology ; G1 Phase ; drug effects ; G2 Phase ; drug effects ; Humans ; Metaphase ; drug effects ; RNA, Small Interfering ; administration & dosage ; pharmacology ; Transfection ; Ubiquitin-Protein Ligases ; administration & dosage ; pharmacology

To investigate the expression of microRNA (miRNA, miR) let-7e-3p in different cervical lesions and its clinical significance.The expression of miR-let-7e-3p in the tissues of normal cervix (=26), high-grade squamous intraepithelial lesion (HSIL) (=37), and cervix carcinoma (=101) were detected by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). The correlation of miR-let-7e-3p expression with the clinicopathological parameters of patients with cervical cancer was analyzed. miR-let-7e-3p mimic was transfected into cervical carcinoma Siha cells. The cell cycle and apoptosis were determined by flow cytometry; cell proliferation was determined by CCK-8 kit; and the migration and invasion of cells were determined by Transwell assay.The relative expression levels of miR-let-7e-3p in normal cervix, HSIL, and cervical carcinoma were 1.45±0.24, 0.79±0.05 and 0.46±0.04, respectively (all<0.05). After transfection with miR-let-7e-3p mimic, the S-phase fraction and apoptosis rate of Siha cells were increased significantly compared with control group[(29.76±6.6)% vs (13.38±1.3)%,<0.05; (5.98±1.38)% vs (3.53±0.79)%,<0.05, respectively]. OD of transfected Siha cells at 48, 72 and 96 h were 0.57±0.11,0.65±0.04 and 0.84±0.14, which were significantly lower than those of untransfected Siha cells (0.74±0.05, 0.93±0.10 and 1.47±0.14, all<0.05). The migration and invasion abilities of transfected Siha cells were not significantly changed (all>0.05).The expression of miR-let-7e-3p is down-regulated in cervical neoplasms, which is associated with cell cycle arrest and proliferation inhibition of cervical cancer cells.
Apoptosis ; drug effects ; genetics ; Carcinoma ; chemistry ; genetics ; Cell Cycle ; drug effects ; genetics ; Cell Line, Tumor ; chemistry ; drug effects ; physiology ; Cell Movement ; drug effects ; genetics ; Cell Proliferation ; drug effects ; genetics ; Cervical Intraepithelial Neoplasia ; chemistry ; genetics ; physiopathology ; Down-Regulation ; physiology ; Female ; Humans ; MicroRNAs ; analysis ; pharmacology ; Neoplasm Invasiveness ; genetics ; physiopathology ; Neoplastic Processes ; Real-Time Polymerase Chain Reaction ; Transfection ; Uterine Cervical Neoplasms ; chemistry ; genetics ; physiopathology

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.
Animals ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; drug effects ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Nanog Homeobox Protein ; genetics ; metabolism ; Octamer Transcription Factor-3 ; genetics ; metabolism ; Patch-Clamp Techniques ; Primary Cell Culture ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Thymosin ; pharmacology

OBJECTIVETo evaluate the effect of Foxo3a gene over-expression on the development of rat ovarian granulosa cells and in prevention of cisplatin-induced ovarian damage in rats.

METHODSRat ovarian granulose cells released mechanically from the ovaries were cultured in vitro and identified with HE staining and immunohistochemical staining for FSHR. A recombinant adenovirus carrying Foxo3a gene was constructed for infecting the granulose cells, and the cell growth and expressions of cyclin D1, p27, Bax, and Bim were detected; the cell apoptosis and cell cycle changes were detected using Hoechst/PI 33342 staining and flow cytometry, respectively. The transfected cells were challenged with cisplatin and the cell apoptosis was detected with flow cytometry.

RESULTSOver 90% of the cultured cells survived and contained more than 95% ovarian granulose cells. Infection of the cells with the recombinant adenovirus resulted in over-expressions of Foxo3a at the mRNA and protein levels at 36 h and 48 h after the infection, respectively. The infected cells showed suppressed proliferation, increased apoptotic rate and cell cycle arrest in G1 phase with increased expressions of Bim, p27, and cyclin D1 but without significant changes in Bax expression. Cisplatin exposure caused a significantly higher apoptosis rate in the infected cells than in the control cells.

CONCLUSIONOver-expression of Foxo3a gene can promote granulose cell apoptosis by increasing Bim expression and cause cell cycle arrest in G1 phase by increasing cyclin D1 and p27 expressions, but can not prevent the toxic effects of cisplatin on ovarian granulosa cells.

Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Bcl-2-Like Protein 11 ; Cell Cycle Checkpoints ; Cell Proliferation ; Cells, Cultured ; Cisplatin ; adverse effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Female ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression ; Granulosa Cells ; cytology ; drug effects ; Membrane Proteins ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Rats ; Transfection ; bcl-2-Associated X Protein ; metabolism