Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Female ; Guanine Nucleotide Exchange Factors ; antagonists & inhibitors ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; MCF-7 Cells ; Maleimides ; pharmacology ; Protein Isoforms ; genetics ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Receptor, ErbB-2 ; genetics ; metabolism ; Tetradecanoylphorbol Acetate ; toxicity

OBJECTIVETo investigate the effects of sphingosine kinase 1 (SphK1) on the proliferation, migration and invasion of human colon cancer LoVo cells, and to explore the related mechanisms.

METHODSHuman colon cancer LoVo cells were divided into three groups: phorbol 12-myristate 13-acetate (PMA) was used to induce the activation of SphK1 in the PMA group, N,N-dimethylsphingosine (DMS) used to suppress the activity of SphK1 in DMS group, and the cells treated with equal amount of 0.9 % NaCl instead of drugs served as the control group. The activity of SphK1 was assayed by autoradiography, the cell proliferation was assessed by MTT assay, cell migration and invasion were examined by Boyden chamber assay, concentrations of sICAM-1 and sVCAM-1 were assayed by ELISA, and RT-PCR and Western blot were used to evaluate the mRNA and protein expression in the cells.

RESULTSThe activity of SphK1 was efficiently induced by PMA and significantly suppressed by DMS. PMA induced cell proliferation in a time- and dose-dependent manner. On the contrast, DMS suppressed cell proliferation in a time- and dose-dependent manner. After treating with PMA, the number of migrating and invasing cells were increased to 143.36 ± 8.73 and 118.46 ± 6.25, significantly higher than those of the control group (75.48 ± 6.12 and 64.19 ± 5.36). After treating with DMS, the number of migrating and invasing cells were decreased to 38.57 ± 3.24 and 32.48 ± 4.27, significantly lower than those of the control group (P < 0.01). The relative expression levels of FAK, ICAM-1 and VCAM-1 mRNA in the PMA group were 0.82 ± 0.06, 0.74 ± 0.05 and 0.89 ± 0.09, and those in the DMS group were 0.23 ± 0.02, 0.26 ± 0.03 and 0.37 ± 0.04, with significant differences between the PMA, DMS and control groups (P < 0.01). Compared with the control group, the relative expression levels of FAK and p-FAK proteins in the PMA group (0.52 ± 0.06 and 0.51 ± 0.06) were significantly elevated, and those of the DMS group (0.20 ± 0.03 and 0.09 ± 0.02) were significantly decreased. In addition, the concentrations of sICAM-1 and sVCAM-1 were significantly elevated with the activation of SphK1. On the contrary, those of the DMS group were significantly reduced with the suppression of SphK1 (Both P < 0.01).

CONCLUSIONSSphK1 may enhance the proliferation, migration and invasion of colon cancer LoVo cells through activating FAK pathway and up-regulating the expression of ICAM-1 and VCAM-1.

Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; enzymology ; metabolism ; pathology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; drug effects ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Sphingosine ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; genetics ; metabolism

OBJECTIVETo investigate the effect of DADLE, a δ-opioid receptor agonist, on the proliferation of human liver cancer HepG2 cells and explore the mechanism involving PKC pathway.

METHODSHepG2 cells were treated with DADLE at different doses (0.01, 0.1, 1.0 and 10 µmol/L). Cell viability was determined using methyl thiazolyl terazolium (MTT) assay. The expression of PKC mRNA and p-PKC protein were examined by RT-PCR and Western blot assay. After treated separately with DADLE plusing NAL or PMA, the cell cycle of HepG2 cells was analyzed by flow cytometer. MTT was used to detect their proliferation capacity and Western blot was used to examine the p-PKC expression. The growth inhibitory rate of HepG2 cells treated with DADLE and cis-diammine dichloridoplatinum (CDDP) was analyzed.

RESULTSDADLE at different concentrations showed an inhibitory effect on the proliferation of HepG2 cells though inhibiting the expression of PKC mRNA and p-PKC protein. The results of flow cytometry showed that compared with the control group, the percentage of S + G(2)/M cells in DADLE-treated group was lowered by 3.94% (P < 0.01). Meanwhile, after treated with NAL and PMA, the percentage was elevated by 3.22% and 3.63%, respectively (P < 0.01). The MTT and Western blot assays showed that compared with the control group, the values of A570 and p-PKC protein levels in the HepG2 cells of DADLE-treated group were significantly decreased (P < 0.01). After treatment with NAL and PMA, the values of A570 and p-PKC protein levels were elevated significantly (P < 0.01). The growth inhibitory rate of DADLE + CDDP group was 79.9%, significantly lower than 25.2% and 43.2% of the DADLE and CDDP groups, respectively.

CONCLUSIONSActivation of δ-opioid receptor by DADLE inhibits the apoptosis of human liver cancer HepG2 cells. The underlying mechanism may be correlated with PKC pathway. DADLE can enhance the chemosensitivity of HepG2 cells to CDDP.

Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Enkephalin, Leucine-2-Alanine ; administration & dosage ; pharmacology ; Hep G2 Cells ; Humans ; Naltrexone ; analogs & derivatives ; pharmacology ; Phosphorylation ; Protein Kinase C ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Opioid, delta ; agonists ; Signal Transduction ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the effect of sphingosine kinase 1 (SphK1) on the proliferation, apoptosis, migration and invasion of colon cancer TH-29 cells and to explore its molecular mechanisms.

METHODSPhorbol 12-myristate 13-acetate (PMA) was used to induce the activity of SphK1 and N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. Cell prolieration and apoptosis were detected by MTT assay and flow cytometry, respectively. The migration and invasion capabilities of the cells were assessed in Transwell chambers. The activity of SphK1 was assayed by autoradiography. Western blot was used to evaluate the protein expression of SphK1, p38, phosphorylated p38 (p-p38) and SAPK/JNK.

RESULTSPMA and DMS were able to induce and suppress the activity and protein expression of SphK1 in a time-dependent manner, respectively. PMA enhanced and DMS suppressed the cell viability in a time- and dose-dependent manner. Being treated with 100 nmol/L PMA or 50 µmol/L DMS for 0, 6, 12, 24 h, the cell apoptosis rates of PMA group were (9.35 ± 0.84)%, (7.61 ± 0.48)%, (5.53 ± 0.76)% and (0.56 ± 0.33)%, contrastly, that of DMS group were (9.18 ± 0.94)%, (12.06 ± 1.41)%, (19.80 ± 2.36)% and (31.85 ± 3.60)%, respectively. Compared with the control group, the cell migration and invasion capabilities of the PMA group were significantly enhanced, and that of the DMS group were significantly suppressed. The migration cell number of control, PMA and DMS groups were 68.75 ± 6.15, 109.33 ± 11.63 and 10.83 ± 2.48, the invasion cell number of control, PMA and DMS groups were 55.42 ± 4.50, 90.58 ± 7.06 and 9.58 ± 2.39, respectively. With the elevating activity and expression of SphK1, the protein expressions of p38, p-p38 and SAPK/JNK were strikingly suppressed. On the contrary, after treating with DMS the protein expressions of p38, p-p38 and SAPK/JNK were enhanced.

CONCLUSIONSSphK1 potently enhances the prolieration, migration and invasion of colon cancer HT-29 cells, meanwhile suppresses the cell apoptosis. The suppressing of the p38 and SAPK/JNK signalling pathways may be one of its molecular mechanisms.

Apoptosis ; drug effects ; Carcinogens ; administration & dosage ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; administration & dosage ; pharmacology ; HT29 Cells ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Neoplasm Invasiveness ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; physiology ; Sphingosine ; administration & dosage ; analogs & derivatives ; pharmacology ; Tetradecanoylphorbol Acetate ; administration & dosage ; analogs & derivatives ; pharmacology ; Time Factors ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVES: Doxycycline is commonly used in medicine for its bacteriostatic antimicrobial properties. Recent studies have reported that doxycycline also has anti-inflammatory effects. Matrix metalloproteinase (MMP)-9 has been found to be involved in the physiological and pathological process of inflammatory airway disease. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, is known to stimulate the expression of MMP and mucin genes in the airway and intestinal epithelial cells. Therefore, the effects and signal pathways of doxycycline on PMA-induced MUC5B expression dependent MMP-9 in human airway epithelial cells were investigated. METHODS: In human NCI-H292 airway epithelial cells, MUC5B and MMP-9 mRNA expression, MUC5B protein expression, and MMP-9 protein activity after the treatment with PMA, MMP-9 or doxycycline were determined by reverse transcriptase-polymerase chain reaction, enzyme immunoassay, gelatin zymography, and Western blot analysis. RESULTS: PMA increased MMP-9 and MUC5B expression. MMP-9 increased MUC5B expression. Doxycycline inhibited PMA-induced MUC5B expression, and PMA-induced MMP-9 mRNA expression and protein activity. Doxycycline inhibited phosphorylation of p38 induced by PMA and MMP-9. CONCLUSION: The results of this study suggest that doxycycline inhibited PMA-induced MUC5B mRNA expression and protein production through the MMP-9 and p38 pathways in human NCI-H292 airway epithelial cells.
Blotting, Western ; Doxycycline ; Epithelial Cells ; Gelatin ; Humans ; Immunoenzyme Techniques ; Inflammation ; Matrix Metalloproteinase 9 ; Mucins ; Phorbols ; Phosphorylation ; Protein Kinase C ; RNA, Messenger ; Signal Transduction ; Tetradecanoylphorbol Acetate ; Thiram

The study was aimed to investigate the effects of protein kinase C (PKC) on standard type CD44 expression and subcellular distribution in human erythrocytes. PKC activity was detected by the incorporation of [gamma-(32)P]-ATP into exogenous substrate, phosphorylation of CD44 was determined by autoradiograph, distribution of CD44 was observed by indirect immunofluorescence, and expression of CD44 was analyzed by flow cytometry. The results showed that PKC activity reached the maximal level at 30 minutes after treatment with phorbol-myristate-acetate (PMA), and the peak of CD44 phosphorylation and CD44 expression appeared at the same time, which all increased significantly as compared with control group (p < 0.001). PKC activation resulted in CD44 aggregation on membrane and colocalization of PKC and CD44. Calphostin C could inhibit the above reaction resulted from PKC activation. It is concluded that PKC activation can up-regulate CD44 expression by phosphorylation, and result in the coherent migration and colocalization of CD44 and PKC in human erythrocytes.
Erythrocyte Count ; Erythrocytes ; enzymology ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Membrane Proteins ; metabolism ; Phosphorylation ; Protein Kinase C ; metabolism ; Tetradecanoylphorbol Acetate ; analogs & derivatives ; Up-Regulation

Arginine vasopressin (AVP), a neurohormone and hemodynamic factor implicated in the pathophysiology of hypertension and congestive heart failure, can also act as a growth-stimulating factor. Our previous work demonstrated that AVP is a mitogen for neonatal rat cardiac fibroblasts (CFs). In the present study, we extended our investigations to adult rat CFs to explore whether AVP could induce adult rat CF proliferation and, if so, to identify the mechanism involved. Adult rat CFs were isolated, cultured and subjected to AVP treatment. DNA synthesis and cell cycle distribution were analyzed by [(3)H]-thymidine incorporation and flow cytometry. Cellular extracellular signal-regulated kinase 1/2 (ERK1/2) activity was measured by in vitro kinase assay using myelin basic protein (MBP) as a substrate. Protein expressions of total- and phospho-ERK1/2, p27(Kip1), cyclins D1, A, E were assessed by Western blot. The results showed that AVP stimulated DNA synthesis in adult rat CFs, and the effect was abolished by a V1 receptor antagonist, d(CH(2))(5)[Tyr(2)(Me), Arg(8)]-vasopressin (0.1 μmol/L), but not by a V2 receptor antagonist, desglycinamide-[d(CH(2))(5), D-Ile(2), Ile(4), Arg8]-vasopressin (0.1 μmol/L). AVP induced an activation of ERK1/2, which could be mimicked by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 30 nmol/L, 5 min), but abolished by depletion of PKC via chronic PMA incubation (2.5 μmol/L, 24 h). In addition, AVP down-regulated protein expression of p27(Kip1), increased protein expressions of cyclins D1, A and E, and induced cell cycle progression from G(0)/G(1) into S stage. Inhibition of ERK1/2 activation by PD98059 (30 μmol/L) abolished the effect of AVP on DNA synthesis, protein expressions of p27(Kip1), cyclins D1, A and E as well as cell cycle progression. These results suggest that AVP is also a growth factor for adult rat CFs. The mitogenic effect of AVP is mediated via V1 receptors and PKC-ERK1/2 pathway. Moreover, AVP modulates the expressions of cell cycle regulatory proteins p27(Kip1) and cyclins D1, A and E, which lie downstream of ERK1/2 activation, and induces cell cycle progression in adult rat CFs.
Animals ; Antidiuretic Hormone Receptor Antagonists ; pharmacology ; Arginine Vasopressin ; pharmacology ; Cell Cycle ; Cell Cycle Proteins ; metabolism ; Cell Proliferation ; Fibroblasts ; cytology ; drug effects ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocardium ; cytology ; Phosphorylation ; Protein Kinase C ; metabolism ; Rats ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo investigate the mechanism of rosiglitazone (RSG, the activator of peroxisome proliferators activated receptor lambda) for inhibiting endothelin-1 (ET-1)-induced neonatal rat cardiac myocyte hypertrophy and the role of protein kinase C (PKC) and c-fos.

METHODSIn vitro cultured neonatal rat cardiac myocytes were treated with ET-1, phorbol ester (PMA, the PKC activator), ET-1+RSG, ET-1+chelerythrine (che, the PKC inhibitor), PMA+RSG, or without treatment (control), respectively. The effects of RSG on the protein content, (3)H-leucine incorporation, PKC activity and C-fos protein expression were observed in the cardiac myocytes stimulated with ET-1 or PMA.

RESULTSAfter two days of culture, the intracellular protein content in ET-1 group and PMA group were increased by 15% (339-/+15 microg/ml) and 13% (329-/+14 microg/ml) as compared with the control cells (290-/+13 microg/ml), respectively (P<0.01). Compared with the ET-1 group, cells treated with ET-1+10(-8) mol/L RSG, ET-1+10(-7) mol/L RSG, and ET-1+che showed decreased intracellular protein content by 10% (303-/+14 microg/ml, P<0.05), 12% (292-/+11 microg/ml, P<0.05), and 13% (291-/+12 microg/ml, P<0.01), respectively. The intracellular protein content in PMA+10(-7) mol/LRSG group was decreased by 10% (P<0.05) in comparison with the PMA group. RSG inhibited protein synthesis enhancement and increased (3)H-leucine incorporation induced by ET-1 and PMA, and antagonized the effects of ET-1 and PMA in promoting PKC activity and c-fos protein expression in the myocytes.

CONCLUSIONThe inhibitory effect of RSG on ET-1- or PMA-induced myocyte hypertrophy is associated with PKC-c-fos pathway.

Animals ; Animals, Newborn ; Blotting, Western ; Cell Enlargement ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelin-1 ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Protein Kinase C ; metabolism ; Proto-Oncogene Proteins c-fos ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Tetradecanoylphorbol Acetate ; pharmacology ; Thiazolidinediones ; pharmacology

OBJECTIVETo observe the effects of signal factors of corticosterone (CS), cAMP, cGMP, Ca2+ andprotein kinase C (PKC) on lymphocyte apoptosis in mouse thymus induced by X-rays of 4 Gy in vitro.

METHODSThe DNA lytic rate for thymocytes was measured by fluorospectrophotometry.

RESULTSThe DNA lyric rate for thymocytes 4-8 hours after irradiation with 2-8 Gy was significantly higher than that in the control (P<0.01). As compared with the control, the DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.05-0.4 microg/mL ionomycin (Iono, P<0.05 or P<0.01) or 0.05-0.4 ng/mL phorbol myristate acetate (PMA, P<0.05 or P<0.01), respectively, was significantly increased, while the rate for thymocytes treated with 50 ng/mL cGMP was not significantly increased. The DNA lytic rate for thymocytes treated with 0.01 micromol/L CS (P<0.01), 50 ng/mL cAMP (P<0.01), 0.2 and 0.4 microg/mL Iono (P<0.05), and 0.2 and 0.4 ng/mL PMA (P<0.05) plus 4-Gy irradiation, respectively, was significantly higher than that treated with single 4-Gy irradiation, while the rate for thymocytes treated with 50 ng/mL cGMP plus 4-Gy irradiation was not increased. When both 0.4 microg/mL Iono and 0.4 ng/mL PMA acted on the thymocytes, the DNA lytic rate for thymocytes was significantly higher than that in the control (P<0.01), the DNA lytic rate for thymocytes treated with both 0.4 microg/mL Iono and 0.4 ng/mL PMA plus 4-Gy irradiation was significantly higher than that treated with single 4-Gy irradiation (P<0.05), but was not significantly higher than that treated with 0.4 microg/mL Iono plus 4-Gy irradiation or 0.4 ng/mL PMA plus 4-Gy irradiation.

CONCLUSIONCS, cAMP, Ca2+, and PKC signal factors can promote thymocyte apoptosis induced by larger dose X-rays.

Animals ; Apoptosis ; drug effects ; radiation effects ; Calcium ; pharmacology ; Corticosterone ; pharmacology ; Cyclic AMP ; pharmacology ; Cyclic GMP ; pharmacology ; Ionomycin ; pharmacology ; Male ; Mice ; Protein Kinase C ; metabolism ; Spectrometry, Fluorescence ; Tetradecanoylphorbol Acetate ; pharmacology ; Thymus Gland ; cytology ; drug effects ; X-Rays

Expression of matrix metalloproteinase-9 (MMP-9) is associated with airway remodeling and tissue injury in asthma. However, little is known about how MMP-9 is up-regulated in airway epithelial cells. In this study, we show that phorbol myristate acetate (PMA) induces MMP-9 expression via a protein kinase Calpha(PKCalpha)-dependent signaling cascade in BEAS-2B human lung epithelial cells. Pretreatment with either GF109203X, a general PKC inhibitor, or Go6976, a PKCalpha/beta isozyme inhibitor, inhibited PMA-induced activation of the MMP-9 promoter, as did transient transfection with PKCalpha antisense oligonuclotides. PMA activated NF-kappaB by phosphorylating IkappaB in these cells and this was also inhibited by GF109203X and Go6976, suggesting that PKCalpha acts as an upstream regulator of NF-kappaB in PMA-induced MMP-9 induction. Our results indicate that a "PKCalpha-NF-kappaB"-dependent cascade is involved in the signaling leading to PMA-induced MMP-9 expression in the lung epithelium.
Up-Regulation/*drug effects ; Tetradecanoylphorbol Acetate/*pharmacology ; Protein Kinase C-alpha/*metabolism ; NF-kappa B/*metabolism ; Matrix Metalloproteinase 9/*metabolism ; Lung/drug effects/*metabolism ; Humans ; Epithelial Cells/drug effects/metabolism ; Cell Line