Expression of protein kinase C-delta (PKC delta) is up-regulated by apoptosis-inducing stimuli. However, very little is known about the signaling pathways that control PKC delta gene transcription. In the present study, we demonstrate that JNK stimulates PKC delta gene expression via c-Jun and ATF2 in response to the anticancer agent doxorubicin (DXR) in mouse lymphocytic leukemia L1210 cells. Luciferase reporter assays showed that DXR-induced activation of the PKC delta promoter was enhanced by ectopic expression of JNK1, c-Jun, or ATF2, whereas it was strongly reduced by expression of dominant negative JNK1 or by treatment with the JNK inhibitor SP600125. Furthermore, point mutations in the core sequence of the c-Jun/ATF2 binding site suppressed DXR-induced activation of the PKC delta promoter. Our results suggest an additional role for a JNK signaling cascade in DXR-induced PKC delta gene expression.
Activating Transcription Factor 2/*physiology ; Animals ; Anthracenes/pharmacology ; Antibiotics, Antineoplastic/*pharmacology ; Apoptosis ; Cell Line, Tumor ; Doxorubicin/*pharmacology ; Mice ; Mitogen-Activated Protein Kinase 8/*physiology ; Mutation ; Promoter Regions, Genetic ; Protein Kinase C-delta/genetics/*metabolism ; Proto-Oncogene Proteins c-jun/antagonists & inhibitors/*physiology ; Signal Transduction/physiology ; Transcription, Genetic

OBJECTIVETo evaluate the effect of PKC-delta inhibitor Rottlerin on human colon cancer cells and its mechanism.

METHODSHuman colon cancer cell line SW1116 cells were treated with Rottlerin. The transcriptional level of DNA methyltransferase (Dnmt)1, Dnmt3a and Dnmt3b was detected by real-time RT-PCR. Cell cycle distribution was evaluated by flow cytometry (FCM). In addition, cellular morphological changes were examined by light microscopy.

RESULTSPKC-delta inhibitor decreased the expression of Dnmt1, Dnmt3a mRNA, up-regulated APC, p21(WAF1) and p16(INK4A) mRNA. Demonstarted by flow cytometry, Rottlerin increased the percentage of cell cycle G0/G1 phase cell numbers (P = 0.02) and decreased the percentage of cell cycle G2/M phase cell numbers (P = 0.01). Remarkable changes of cellular morphology were observed under light microscope: The volume and cytoplasm of cells treated with Rottlerin were increased. The cell contour was not very clear, and mitotic figures were less frequently seen.

CONCLUSIONPKC-delta inhibitor Rottlerin inhibites cell division and proliferation of the colon cancer SW1116 cells through regulating DNA methylation and blocking the signaling pathway of mitogen-activated protein kinase (MAPK).

Acetophenones ; pharmacology ; Adenomatous Polyposis Coli Protein ; genetics ; Benzopyrans ; pharmacology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Protein Kinase C-delta ; antagonists & inhibitors ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects

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 &delta;-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