OBJECTIVE: To investigate the effects of paclitaxel, quizartinib and their combination on proliferation, apoptosis and FLT3/STAT5 pathway of human leukemia cell line MV4-11 (FLT3-ITD+). METHODS: MV4-11 cells were treated with paclitaxel and quizartinib at different concentrations for 24 h, 48 h and 72 h, respectively, and then the two drugs were combined at 48 h to compare the inhibition of proliferation, the apoptosis rate was detected by flow cytometry, the expression of FLT3 and STAT5 mRNA was determined by fluorescence quantitative PCR, and the protein expression of FLT3, p-FLT3, STAT5 and p-STAT5 was determined by Western blot. RESULTS: Different combination groups of paclitaxel and quizartinib had synergistic inhibitory effect. The cell survival rate in the combination group was significantly lower than that in the single drug group (P<0.05). The cell apoptosis rate in the combination group was significantly higher than that in the single drug group (P<0.001). The expression of FLT3 mRNA in combination group was significantly higher than that in two single drugs (P<0.01). The expression of STAT5 mRNA in combination group was significantly higher than that in quizartinib group (P<0.001); increased compared with paclitaxel group, but there was no statistical significance. The expression level of p-FLT3、p-STAT5 protein in the combination group was significantly lower than that in the single drug group (P<0.05, P<0.05). CONCLUSION Paclitaxel combined with quizartinib can synergistically inhibit the proliferation of MV4-11 cell line and promote the apoptosis of MV4-11 cell line by inhibiting the activity of FLT3/STAT5 pathway.
Apoptosis ; Benzothiazoles ; Cell Line, Tumor ; Humans ; Leukemia, Myeloid, Acute/genetics* ; Paclitaxel/therapeutic use* ; Phenylurea Compounds ; RNA, Messenger ; STAT5 Transcription Factor/pharmacology* ; Signal Transduction ; fms-Like Tyrosine Kinase 3

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.
2-Aminoadipic Acid ; toxicity ; Animals ; Anti-Inflammatory Agents ; therapeutic use ; Astrocytes ; pathology ; Carbenoxolone ; pharmacology ; Caspase 6 ; deficiency ; metabolism ; Connexin 43 ; metabolism ; Disease Models, Animal ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Female ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Mice ; Mice, Transgenic ; Microglia ; pathology ; Minocycline ; therapeutic use ; Neuralgia ; chemically induced ; drug therapy ; pathology ; Pain Measurement ; Phenylurea Compounds ; pharmacology ; Sex Characteristics ; Spinal Cord ; pathology ; Time Factors

Objective: To explore the effects and possible mechanisms of the novel pan-FGFR inhibitor BGJ398 on KG-1 cells in vitro. Methods: Effects of BGJ398 on cells proliferation were detected by CCK-8, the apoptosis was assessed by Annexin V-FITC. Reverse transcriptionquantitative polymerase chain reaction (q-PCR) analysis was used to detect the expression of apoptosis-related genes B cell lymphoma-2 (Bcl-2) and caspase-3. Western blotting analysis was performed to explore the proteins expression levels of Bcl-2, caspase-3 and the expression of p-AKT, p-S6K, p-ERK and FGFR1. Results: BGJ398 effectively inhibited cell proliferation by dose-dependent manners. BGJ398(1.4 µmol/L) induced apoptosis of KG-1 cells by 36.4%, compared with 4.5% in the control group(P<0.001). Treatment with BGJ398 at 1.4 µmol/L led to significant increases in the expression levels of caspase-3, and decreases in the expression of Bcl-2 (P<0.005). In accordance with these results, Western blot analysis further confirmed the increased expression of Bcl-2 protein along with elevated caspase-3 activity. In addition, BGJ398 markedly down-regulated FGFR1OP2-FGFR1 fusion protein, p-AKT and p-S6K expression, but not p-ERK expression. Conclusion: Novel pan-FGFR inhibitor BGJ398 substantially suppressed KG-1 cell growth and induced apoptosis by inhibiting the expression of FGFR1, p-AKT, p-S6K and regulating apoptosis-related proteins.
Apoptosis ; Caspase 3 ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Phenylurea Compounds/pharmacology* ; Protein Kinase Inhibitors/pharmacology* ; Pyrimidines/pharmacology*

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

Neurofibrillary pathology of abnormally hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD) and other tauopathies. Phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase-3 beta (GSK-3β) signaling pathway is pivotal for tau phosphorylation. Inhibition of soluble epoxide hydrolase (sEH) metabolism has been shown to effectively increase the accumulation of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid and have been demonstrated to have neuroprotective effects. However, little is known about the role of sEH in tau phosphorylation. The present study investigated the role of a sEH inhibitor, 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl] urea (TPPU), on HO-induced tau phosphorylation and the underlying signaling pathway in human embryonic kidney 293 (HEK293)/Tau cells. We found that the cell viability was increased after TPPU treatment compared to control in oxidative stress. Western blotting and immunofluorescence results showed that the levels of phosphorylated tau at Thr231 and Ser396 sites were increased in HO-treated cells but dropped to normal levels after TPPU administration. HOinduced an obvious decreased phosphorylation of GSK-3β at Ser9, an inactive form of GSK-3β, while there were no changes of phosphorylation of GSK-3β at Tyr216. TPPU pretreatment maintained GSK-3β Ser 9 phosphorylation. Moreover, Western blotting results showed that TPPU upregulated the expression of p-Akt. The protective effects of TPPU were found to be inhibited by wortmannin (WT, a specific PI3K inhibitor). In conclusion, these results suggested that the protective effect of TPPU on HO-induced oxidative stress is associated with PI3K/Akt/GSK-3β pathway.
Cell Survival ; drug effects ; Enzyme Inhibitors ; pharmacology ; Glycogen Synthase Kinase 3 beta ; metabolism ; HEK293 Cells ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidative Stress ; Phenylurea Compounds ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Piperidines ; pharmacology ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; tau Proteins ; metabolism

BACKGROUND/AIMS: Silibinin, the main component of silymarin, is used as a hepatoprotectant and exhibits anticancer effects against various cancer cells. This study evaluated the effects of a combination of silibinin with either gefitinib or sorafenib on hepatocellular carcinoma (HCC) cells. METHODS: Several different human HCC cell lines were used to test the growth-inhibiting effects and cell toxicity of silibinin both alone and in combination with either gefitinib or sorafenib. The cell viability and growth inhibition were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, trypan blue staining, and a colony-forming assay. Furthermore, changes in epidermal growth factor receptor (EGFR)-related signals were evaluated by Western blot analysis. RESULTS: Gefitinib, sorafenib, and silibinin individually exhibited dose-dependent antiproliferative effects on HCC cells. Combined treatment with silibinin enhanced the gefitinib-induced growth-inhibiting effects in some HCC cell lines. The combination effect of gefitinib and silibinin was synergistic in the SNU761 cell line, but was only additive in the Huh-BAT cell line. The combination effect may be attributable to inhibition of EGFR-dependent Akt signaling. Enhanced growth-inhibiting effects were also observed in HCC cells treated with a combination of sorafenib and silibinin. CONCLUSIONS: Combined treatment with silibinin enhanced the growth-inhibiting effects of both gefitinib and sorafenib. Therefore, the combination of silibinin with either sorafenib or gefitinib could be a useful treatment approach for HCC in the future.
Antineoplastic Agents/*pharmacology ; Carcinoma, Hepatocellular/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cell Survival/drug effects ; Down-Regulation/drug effects ; Drug Screening Assays, Antitumor ; Drug Synergism ; Humans ; Liver Neoplasms/metabolism/pathology ; Niacinamide/*analogs & derivatives/pharmacology ; Phenylurea Compounds/*pharmacology ; Proto-Oncogene Proteins c-akt/metabolism ; Quinazolines/*pharmacology ; Receptor, Epidermal Growth Factor/metabolism ; Signal Transduction/drug effects ; Silymarin/*pharmacology

OBJECTIVETo investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402.

METHODSSRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib. Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins.

RESULTSSRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib, with a combination index of <0.9. The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5μmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively(P<0.05). Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP, Caspase-8 and Caspase-3, and promoted the expression levels of p53, p21 and γ-H2AX significantly.

CONCLUSIONSN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis.

Apoptosis ; Camptothecin ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Histones ; metabolism ; Humans ; Liver Neoplasms ; pathology ; Niacinamide ; analogs & derivatives ; pharmacology ; Phenylurea Compounds ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.
Naphthaleneacetic Acids ; pharmacology ; Phenylurea Compounds ; pharmacology ; Plant Growth Regulators ; pharmacology ; Plant Shoots ; drug effects ; growth & development ; Plant Stems ; drug effects ; growth & development ; Seedlings ; drug effects ; growth & development ; Thiadiazoles ; pharmacology ; Tissue Culture Techniques ; Tulipa ; drug effects ; growth & development

The effect of plant growth regulator forchlorfenuron (CPPU) 1 x 10(-6), 0.67 x 10(-6), 0.5 x 10(-6) on fruit morphology and effective components lignans was studied. Those morphologies were the combination of four basic morphological changes. The result showed, diametre were increased and longitudinal diametre of fruits were inhibited by foliage fertilizers including CPPU. At the same time, 1 000-grain weight and yield showed the varying degrees increase under CPPU. The order of the degree was 0.5 x 10(-6) > 1 x 10(-6) > 0.67 x 10(-6). Six lignans content of Schisandra chinensis of different harvest time and different CPPU processing groups were determined, the results showed that lignans accumulation occurred mainly in periods of premature the half mature fruiting stages. Under the 0.67 x 10(-6) CPPU treatment, schisandrol B, schisandrin B, schisandrin C content of S. chinensis showed different increase.
Chromatography, High Pressure Liquid ; Cyclooctanes ; analysis ; metabolism ; Dioxoles ; analysis ; metabolism ; Dose-Response Relationship, Drug ; Fruit ; drug effects ; growth & development ; metabolism ; Lignans ; analysis ; metabolism ; Phenylurea Compounds ; pharmacology ; Polycyclic Compounds ; analysis ; metabolism ; Pyridines ; pharmacology

This study was aimed to investigate the effects of sorafenib on proliferation and apoptosis of MM cell line RPMI-8226, and to explore the its potential anti-tumor mechanism. The inhibitory rate of multiple myeloma cell proliferation was tested by MTT. Transmission electron microscopy was used to observe morphological and ultrastructural changes of RPMI-8226 cells treated with sorafenib. The effects of sorafenib on the apoptosis and cell cycle of RPMI-8226 cells was detected by flow cytometry. The effects of sorafenib on the expression of caspase-3, BCL-2 and MCL-1 mRNA and protein were assayed by RT-PCR and Western blot respectively. The results showed that sorafenib (0-10.0 µmol/L) could obviously inhibit the proliferation of RPMI-8226 cells in time and dose-dependent manner. Flow cytometry results showed that sorafenib could induce apoptosis of RPMI-8226 cells, the difference was statistical significance (P < 0.05). Sorafenib mainly arrested RPMI-8226 cells in the G1 phase (P < 0.05). Typical apoptotic morphological and ultrastructural changes of MM cells could be observed under transmission electron microscope, Examination of cellular signaling pathways showed that sorafenib induced upregulation of cleaved-caspase-3 expression, and simultaneous downregulation of BCL-2 and MCL-1 expression. It is concluded that sorafenib displays anti-myeloma activity. Activating the death receptor pathway and arresting cell cycle may be two of the relatated mechanisms.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Multiple Myeloma ; drug therapy ; pathology ; Niacinamide ; analogs & derivatives ; pharmacology ; Phenylurea Compounds ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; Signal Transduction