OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

OBJECTIVE: To investigate the effect of danusertib (Danu), an inhibitor of Aurora kinase, on the proliferation, cell cycle, apoptosis, and autophagy of hepatocellular carcinoma HepG2 cells and explore the underlying mechanisms. METHODS: MTT assay was used to examine the effect of Danu on the viability of HepG2 cells to determine the IC50 of Danu. The effect of Danu on cell cycle distribution, apoptosis and autophagy were determined using flow cytometry. Western blotting was used to detect the expressions of the proteins related to cell cycle, apoptosis and autophagy. Chloroquine was used to suppress Danuinduced autophagy to test the apoptosis-inducing effect of Danu. RESULTS: Danu significantly inhibited the proliferation of HepG2 cells with IC of 39.4 μmol and 14.4 μmol at 24 h and 48 h, respectively. Danu caused cell cycle arrest in G/M phase in HepG2 cells and led to polyploidy accumulation via up-regulating the expressions of p53 and p21 and down-regulating the expressions of cyclin B1 and DC2. Danu also caused apoptosis of HepG2 cells through up-regulating the expressions of Bax, Puma, cleaved caspase-3, cleaved caspase-9, cleaved PARP and cytochrome C and down-regulating the expressions of Bcl-xl and Bcl-2. Danu induced autophagy via activating AMPK signaling and inhibiting PI3K/PTEN/AKT/mTOR axis, and inhibition of Danu-induced autophagy with chloroquine enhanced the pro-apoptotic effect of Danu. CONCLUSIONS Danu inhibits cell proliferation and induces cell cycle arrest in G/M phase, apoptosis and cytoprotective autophagy in HepG2 cells.
Apoptosis ; drug effects ; Autophagy ; drug effects ; Benzamides ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Neoplasm Proteins ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Pyrazoles ; pharmacology

This study was aimed to investigate the effects of blockade of Ca(2+) activated channel KCa3.1 and voltage-gated potassium channel Kv1.3 of the monocytes/macrophages on inflammatory monocyte chemotaxis. Chemotaxis assay was used to test the inflammatory Ly-6C(hi) monocyte chemotaxis caused by the monocytes/macrophages. The proliferation of monocytes/macrophages was detected by cell counting kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was applied to detect the C-C motif ligand 7 (CCL7) in cultured media. The results showed that the recruitment of Ly-6C(hi) monocyte induced by monocytes/macrophages was suppressed by the potent Kv1.3 blocker Stichodactyla helianthus neurotoxin (ShK) or the specific KCa3.1 inhibitor TRAM-34. Meanwhile, the proliferation of monocytes/macrophages was significantly inhibited by ShK. The response of Ly-6C(hi) monocyte pretreated with ShK or TRAM-34 to CCL2 was declined. These results suggest that KCa3.1 and Kv1.3 may play an important role in monocytes/macrophages' proliferation and migration.
Cell Movement ; Cell Proliferation ; Cnidarian Venoms ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Humans ; Kv1.3 Potassium Channel ; antagonists & inhibitors ; physiology ; Macrophages ; cytology ; Monocytes ; cytology ; Protein Structure, Tertiary ; Pyrazoles ; pharmacology ; Small-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors ; physiology

OBJECTIVETo explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell (HUVEC).

METHODSCells were treated with 40 Μmol/L of the ppo3a, ppo3b, ppo3i, and 0.1% DMSO (control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein (HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B (SRB) assay.

RESULTSPpo3a, ppo3b, and ppo3i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3a, ppo3b, and ppo3i.

CONCLUSIONSppo3a, ppo3b, and ppo3i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

Apoptosis ; drug effects ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP70 Heat-Shock Proteins ; analysis ; physiology ; Humans ; Pyrazoles ; pharmacology ; Tumor Suppressor Protein p53 ; analysis ; physiology

Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.
Apoptosis ; drug effects ; Cell Line, Tumor ; Enzyme Activation ; drug effects ; Humans ; Ligands ; Molecular Docking Simulation ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; genetics ; metabolism ; Oximes ; metabolism ; pharmacology ; Protein Conformation ; Proto-Oncogene Proteins c-akt ; metabolism ; Pyrazoles ; metabolism ; pharmacology ; Retinoid X Receptor alpha ; chemistry ; genetics ; metabolism ; Thiazoles ; metabolism ; pharmacology ; Transcription, Genetic ; drug effects ; Transcriptional Activation ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.
Amides ; pharmacology ; Animals ; Butyric Acid ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Extraembryonic Membranes ; cytology ; drug effects ; Folic Acid ; pharmacology ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Kruppel-Like Transcription Factors ; metabolism ; Mice ; Octamer Transcription Factor-3 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; SOXB1 Transcription Factors ; metabolism ; Thiocarbamates ; pharmacology ; Thiosemicarbazones

OBJECTIVETo investigate the proliferation inhibitory role and mechanism of PI3Kδ inhibitor CAL-101 on multiple myeloma (MM) cells, and to provide new therapeutic options for MM treatment.

METHODSMM cell lines U266 and RPMI8226 cells were treated with various concentrations of CAL-101. MTT assay and CalcuSyn software were performed to determine the inhibitory effect of CAL-101 and the synergistic effect with PCI- 32765, SAHA (suberoylanilide hydroxamic acid), BTZ (Bortezomib) on MM cells. The protein expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ processed by CAL-101 were analyzed by Western blot.

RESULTSCAL-101 at concentration of 15, 20, 25, 30 and 40 μmol/L could induce significant dose-dependent proliferation inhibition on U266 cells after treatment for 48 hours. The cell proliferation inhibition rates were (33.54 ± 1.23)%, (41.72 ± 1.78)%, (53.67 ± 2.01)%, (68.97 ± 2.11)% and (79.25 ± 1.92)%, respectively. Similar results were found in RPMI8226 cell line. Western blots showed high expression level of p-AKT, p-ERK, AKT, ERK and PI3Kδ in cell lines and MM primary cells. p-AKT and p-ERK protein expression levels were down-regulated significantly by CAL-101 treatment. Synergistic effect has been verified between CAL-101 and PCI-32765, SAHA and Bortezomib in U266 cell line, and PCI-32765, Bortezomib in RPMI8226 cell line with CI values less than 1.

CONCLUSIONCAL-101 could inhibit proliferation of MM cell lines. High levels of p-AKT, p-ERK, AKT, ERK and PI3Kδ protein expression were observed in both cell lines and primary cells. Down-regulation of p-AKT and p-ERK probably related with the mechanism of CAL-101 in MM cell proliferation inhibition. CAL-101 has significant synergistic effect with PCI-32765, SAHA and BTZ.

Boronic Acids ; Bortezomib ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Multiple Myeloma ; pathology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Protein Kinase Inhibitors ; pharmacology ; Purines ; pharmacology ; Pyrazines ; Pyrazoles ; Pyrimidines ; Quinazolinones ; pharmacology

Animals ; Cannabinoid Receptor Antagonists ; therapeutic use ; Cannabinoids ; pharmacology ; Fibrosis ; metabolism ; Humans ; Liver Cirrhosis ; etiology ; metabolism ; therapy ; Piperidines ; therapeutic use ; Pyrazoles ; therapeutic use ; Receptor, Cannabinoid, CB1 ; metabolism ; Receptor, Cannabinoid, CB2 ; metabolism ; Receptors, Cannabinoid ; metabolism ; Scleroderma, Diffuse ; metabolism ; Signal Transduction ; drug effects ; Skin ; metabolism ; Smad Proteins ; metabolism ; Transforming Growth Factor beta1 ; metabolism

This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.
Apoptosis ; drug effects ; Celecoxib ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclooxygenase 2 ; metabolism ; Cyclooxygenase 2 Inhibitors ; pharmacology ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Oncogene Proteins ; metabolism ; Pyrazoles ; pharmacology ; Sulfonamides ; pharmacology

OBJECTIVETo investigate whether cannabinoid receptor 1 (CB1R) is involved in nerve growth in endometriosis-associated ectopic cyst.

METHODSThe effect of CB1R agonist and antagonist on the expression of pan-neuronal marker protein gene product (PGP) 9.5 in ectopic cyst was examined by immunofluorescence and Western blot in endometriosis model of 18 rats.

RESULTSImmunofluorescence revealed that PGP 9.5 was expressed in the nerve fibers and was mainly distributed in the cyst hilum. Western blot revealed that the protein density of either PGP 9.5 (2 week: 0.38 ± 0.05; 4 week: 0.63 ± 0.03; 8 week: 0.80 ± 0.07, P < 0.01) or CB1R (2 week: 0.48 ± 0.04; 4 week: 0.68 ± 0.01; 8 week: 0.80 ± 0.03, P < 0.01) in the ectopic cyst increased with cyst size. In addition, compared to control group (0.75 ± 0.01), PGP 9.5 expression in the ectopic cyst was promoted by CB1R agonist ACPA (0.81 ± 0.01, P < 0.05), and inhibited by CB1R antagonist AM251 (0.67 ± 0.03, P < 0.01).

CONCLUSIONSCB1R was involved in the nerve growth of ectopic cyst associated with endometriosis.

Animals ; Blotting, Western ; Cysts ; metabolism ; Disease Models, Animal ; Endometriosis ; metabolism ; Female ; Peripheral Nerves ; growth & development ; metabolism ; Piperidines ; pharmacology ; Pyrazoles ; pharmacology ; Rats ; Receptor, Cannabinoid, CB1 ; antagonists & inhibitors ; physiology ; Ubiquitin Thiolesterase ; metabolism