OBJECTIVETo investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells.

METHODSCLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry.

RESULTSCLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01).

CONCLUSIONCLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.

ATP Binding Cassette Transporter, Sub-Family B ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Survival ; Clusterin ; genetics ; metabolism ; Down-Regulation ; Doxorubicin ; Drug Resistance, Neoplasm ; Humans ; Liver Neoplasms ; metabolism ; pathology ; RNA, Small Interfering ; genetics ; Transfection

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3beta. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3beta. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3beta phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3beta signaling mediates anti-apoptotic effect of clusterin.
Animals ; *Apoptosis ; Blotting, Western ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Line ; Chromones/pharmacology ; Clusterin/metabolism/*pharmacology ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Hydrogen Peroxide/pharmacology ; LDL-Receptor Related Protein 2/metabolism ; Morpholines/pharmacology ; Myocytes, Cardiac/*metabolism ; *Oxidative Stress ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; RNA, Small Interfering ; Rats ; Reactive Oxygen Species/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; *Signal Transduction/drug effects

In the present study, we investigated whether and how the mineralocorticoid receptor antagonist spironolactone affects cardiac growth and development through apoptosis and cell proliferation in the neonatal rat heart. Newborn rat pups were treated with spironolactone (200 mg/kg/d) for 7 days. The cell proliferation was studied by PCNA immunostaining. The treatment with spironolactone decreased proliferating myocytes by 32% (P<0.05), and reduced myocytes apoptosis by 29% (P<0.05). Immunoblot and immunohistochemistry for the expression of p38, p53, clusterin, TGF-beta2, and extracellular signal-regulated kinase were performed. In the spironolactone group, p38, p53, clusterin, and TGF-beta2 protein expression was significantly decreased (P<0.05). These results indicate that aldosterone inhibition in the developing rat heart induces cardiac growth impairment by decreasing proliferation and apoptosis of myocytes.
Aldosterone Antagonists/*pharmacology ; Animals ; Animals, Newborn ; *Apoptosis ; Cell Proliferation ; Clusterin/genetics/metabolism ; Female ; Heart/*drug effects/growth & development ; Proliferating Cell Nuclear Antigen/metabolism ; Rats ; Rats, Sprague-Dawley ; Spironolactone/*pharmacology ; Transforming Growth Factor beta2/genetics/metabolism ; Tumor Suppressor Protein p53/genetics/metabolism ; p38 Mitogen-Activated Protein Kinases/genetics/metabolism