OBJECTIVE: To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. METHODS: Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. RESULTS: Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P < 0.01) and decreased levels of Bcl-2 (P < 0.05) and ACAN (P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P < 0.05). CONCLUSION Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.
Animals ; Apoptosis ; Caspase 3/metabolism* ; Cells, Cultured ; Chondrocytes ; Dinoprostone/pharmacology* ; Disease Models, Animal ; Indoles ; Inflammation/drug therapy* ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Mice ; Mice, Inbred C57BL ; Osteoarthritis/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; RNA, Small Interfering/metabolism* ; Rats ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

The endophytic fungus Nigrospora sphaerica S5 derived from the semi-mangrove plant Myoporum bontioides was fermented. Its metabolites were purified by column chromatography. Nine compounds were obtained and identified as terezine P(1), 3-(1-hydroxyethyl)-4-methyl dihydrofuran-2(3H)-one(2), methylhydroheptelidate(3), hydroheptelidic acid(4), 5, 7-dimethoxy-4, 6-dimethylphthalide(5),(3R,4S)-(-)-4-hydroxymellein(6), pestalopyrone(7), indole-3-formaldehyde(8) and p-hydroxybenzaldehyde(9) by spectroscopic techniques. Terezine P(1) was a new alkaloid belonging to the terezine class with a pyrazine ring. Compounds 2-7 were lactones, of which 3 and 4 belonged to sesquiterpenes. Compounds 8 and 9 were indole alkaloids and phenols, respectively. Compounds 3-6 were purified from Nigrospora sp. for the first time. These compounds showed different degrees of antibacterial activity against Staphylococcus aureus, Escherichia coli of O6 serotype and E. coli of O78 serotype.
Alkaloids ; Anti-Bacterial Agents/pharmacology* ; Ascomycota/chemistry* ; Escherichia coli ; Formaldehyde ; Indoles/pharmacology* ; Lactones ; Molecular Structure ; Myoporum/microbiology* ; Phenols ; Pyrazines ; Sesquiterpenes

OBJECTIVES: Lung cancer is one of the most common malignant tumors in the world, and its lethality ranks the first among many malignant tumors. For non-small cell lung cancer (NSCLC) patients, due to the high mortality rate, the overall 5-year survival rate is less than 15%. When NSCLC undergoes local invasion, the 5-year survival rate is only 20%, and it is even lower when distant metastasis occurs up to 4%. Almonertinib is an innovative drug independently researched and developed by China with independent intellectual property rights. As an epidermal growth factor receptor tyrosine kinase inhibitor, almonertinib is mainly used for locally advanced or metastatic NSCLC patients with epidermal growth factor receptor (EGFR) T790M mutation. This study aims to investigate the effects of almonertinib on the proliferation, invasion and migration of NSCLC cells in vitro. METHODS: NSCLC cells H1975 and PC-9 were cultured in vitro. The effects of almonertinib on the proliferation, apoptosis, invasion, and migration of H1975 and PC-9 cells were detected by CCK-8 assay, apoptotic assay and Transwell assay. The expression of invasion and migration related proteins was detected by Western blotting. RESULTS: The CCK-8 experiment showed that almonertinib inhibited the proliferation of H1975 and PC-9 cells in a time- and dose-dependent manner. The IC CONCLUSIONS Almonertinib can inhibit the proliferation, invasion, and migration of NSCLCH1975 and PC-9 cells in vitro and vivo, and promote the apoptosis of H1975 and PC-9 cells. The underlying mechanism may be related to the inhibition of tumor cell epithelial mesenchymal transformation and metalloproteinase expression.
Acrylamides ; Animals ; Apoptosis ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; ErbB Receptors/genetics* ; Humans ; Indoles ; Lung Neoplasms ; Mice ; Mice, Nude ; Mutation ; Protein Kinase Inhibitors/pharmacology* ; Pyrimidines

According to drug design flattening principle,a series of novel indole podophyllotoxin derivatives which were introduced different indole substituents in C-4 position on the basis of podophyllotoxin nucleus were synthesized with the starting material podophyllotoxin and 1 H-indole-5-carboxylic acid. Its anti-tumor activity in vitro was tested in order to screen for high-efficiency and low-toxic compounds. Six target compounds were synthesized,and were confirmed by~1 H-NMR,~(13)C-NMR,HR-ESI-MS and melting point determination analysis. All these target compounds were not reported by previous literature. Using etoposide as positive control drug,all the target compounds were screened for cytotoxicity against He La cells,K562 cells and K562/A02 cell in vitro by MTT method. The antitumor activity screening results showed that compounds 4 b,4 e,4 f exhibited higher inhibitory rate against He La cells and K562 cells than those of control drug VP-16. This route has the advantages on simple operation and reasonable design,provides some practical reference value for the further development on the structure modification of podophyllotoxin and study on anti-tumor activity.
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Drug Screening Assays, Antitumor ; HeLa Cells ; Humans ; Indoles ; chemical synthesis ; pharmacology ; K562 Cells ; Podophyllotoxin ; chemical synthesis ; pharmacology ; Structure-Activity Relationship

Two new isomeric modified tripeptides, aspergillamides C and D (compounds 1 and 2), together with fifteen known compounds (compounds 3-17), were obtained from the marine sponge-derived fungus Aspergillus terreus SCSIO 41008. The structures of the new compounds, including absolute configurations, were determined by extensive analyses of spectroscopic data (NMR, MS, UV, and IR) and comparisons between the calculated and experimental electronic circular dichroism (ECD) spectra. Butyrolactone I (compound 11) exhibited strong inhibitory effects against Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) with the IC being 5.11 ± 0.53 μmol·L, and acted as a noncompetitive inhibitor based on kinetic analysis.
4-Butyrolactone ; analogs & derivatives ; chemistry ; isolation & purification ; pharmacology ; Animals ; Aspergillus ; chemistry ; Chemistry Techniques, Analytical ; Dipeptides ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Indoles ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Mycobacterium tuberculosis ; drug effects ; Peptides ; chemistry ; isolation & purification ; pharmacology ; Polyketides ; chemistry ; isolation & purification ; pharmacology ; Porifera ; microbiology ; Protein Tyrosine Phosphatases ; chemistry

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

OBJECTIVETo investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells.

METHODSMC-3T3E1 cells were seeded in 96-well plates at the density of 1.5×10(4) cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-28), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34)+1 µmol/L Go6983, 1 µmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells.

RESULTSCCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05); this inhibitory effect was blocked by Go6983 (P<0.05).

CONCLUSIONs A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.

3T3 Cells ; Animals ; Apoptosis ; Cell Proliferation ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Mice ; Osteoblasts ; Parathyroid Hormone ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Signal Transduction

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

OBJECTIVETo explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC).

METHODSBy using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test.

RESULTSThe effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05).

CONCLUSIONThe rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.

Cell Culture Techniques ; Cell Separation ; Flow Cytometry ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Indoles ; pharmacology ; Pyrimidines ; pharmacology

To investigate the effect of a novel EZH2 inhibitor GSK126 on cell growth, apoptosis and migration of prostate cancer cells.Prostate cancer PC-3 and DU145 cells were treated with GSK126 at different doses. Cell growth was detected by sulforhodamine assay. Cell apoptosis was assayed by Annexin V-/PI kit. Transwell chamber and wound healing assays were conducted to detect cell migration. The mRNA level was detected by quantitative PCR, and protein expression was detected by Western blot analysis.GSK126 showed significant effect on cell growth and apoptosis when the dose was higher than 50 μmol/L. Wound healing assay revealed that scratch space in PC-3 cells was significantly increased in a dose-dependent manner in GSK126-treated groups[(247.2±24.4),(347.2±19.2) and (410.5±18.1) μm in low, medium and high dose (5.0, 20.0, 50.0 μmol/L), respectively] as compared with the control group[(171.3±17.8) μm](all<0.05). Transwell assay showed that migrated PC-3 cells in control group was 322.0±17.9,while those in GSK126-treated groups were 198.3±15.4 (low),82.7±6.2 (medium) and 30.2±4.1 (high), and the differences between the control group and GSK126-treated groups were significant(all<0.05). In addition, GSK126 up-regulated E-cadherin mRNA expression and down-regulated N-cadherin and Vimentin mRNA expression, whereas had no significant effect on Snail, Fibronectin and VEGF-A mRNA expression. The protein expression of E-cadherin was elevated but VEGF-A protein did not change in GSK126-treated groups. Similar results were exhibited in DU145 cell.GSK126 can significantly inhibit cell migration and invasion in prostate cancer PC-3 and DU145 cells, which may be resulted from its effect on epithelial-mesenchymal transition. GSK126 may be used as a potential anti-prostate cancer dug in clinic.
Apoptosis ; drug effects ; Cadherins ; analysis ; drug effects ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Drug Screening Assays, Antitumor ; methods ; Enhancer of Zeste Homolog 2 Protein ; analysis ; drug effects ; metabolism ; Fibronectins ; analysis ; drug effects ; metabolism ; Humans ; Indoles ; pharmacology ; Male ; Prostatic Neoplasms ; chemistry ; genetics ; physiopathology ; Pyridones ; pharmacology ; RNA, Messenger ; Up-Regulation ; drug effects ; Vascular Endothelial Growth Factor A ; analysis ; drug effects ; Vimentin ; analysis ; drug effects ; metabolism