Zhengyu Wei

G Protein–Coupled Receptor Kinase 2 Activity Impairs Cardiac Glucose Uptake and Promotes Insulin Resistance After Myocardial Ischemia

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the &bgr;-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through &bgr;-adrenergic receptors and metabolism through the insulin receptor.Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile.

Gefitinib resistance in HCC mahlavu cells: Upregulation of CD133 expression, activation of IGF‐1R signaling pathway, and enhancement of IGF‐1R nuclear translocation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer which accounts for more than half million deaths annually worldwide. While the incidence of HCC is still on the rise, options of treatment are limited and the overall survival rate is poor. The acquisition of cancer drug resistance remains one of the key hurdles to successful treatment. Clearly, a thorough understanding of the underlying mechanisms is needed for new strategies to design novel treatments and/or to improve the current therapies. In the present study, we examined the expression of cancer stem cell (CSC) marker CD133, the activation of insulin‐like growth factor 1 receptor (IGF‐1R) signaling, and the nuclear translocation of IGF‐1R in HCC Mahlavu cells under the treatment of gefitinib, a cancer drug that inhibits epidermal growth factor receptor (EGFR) pathway. Our results demonstrated that Mahlavu cells exhibited strong gefitinib resistance and the CD133 expression level was dramatically increased (from 3.88% to 32%) after drug treatment. In addition, the gefitinib treated cells displayed increased levels of phosphorylation in IGF‐1R and Akt, indicating the intensified activation of this cancer‐associated signaling pathway. Moreover, we revealed that IGF‐1R underwent nuclear translocation in gefitinib treated cells using confocal microscopy. The IGF‐1R nuclear translocation was enhanced under gefitinib treatment and appeared in a dose‐dependent manner. Our findings suggest that increased IGF‐1R nuclear translocation after gefitinib treatment may contribute to the drug resistance and IGF1‐R activation, which might also associate with the upregulation of CD133 expression. J. Cell. Physiol. 227: 2947–2952, 2012.

Targeted Therapies in the Treatment of Advanced Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and the third leading cause of cancer death. It has been a major worldwide health problem with more new cases being diagnosed each year. The current available therapies for patients with advanced HCC are extremely limited. Therefore, it is of great clinical interests to develop more effective therapies for systemic treatment of advanced HCC. Several promising target-based drugs have been tested in a number of clinical trials. One breakthrough of these efforts is the approved clinical use of sorafenib in patients with advanced HCC. Targeted therapies are becoming an attractive option for the treatment of advanced HCC. In this review, we summarize the most recent progress in clinical targeted treatments of advanced HCC.

Growth Inhibition of Human Hepatocellular Carcinoma Cells by Overexpression of G-Protein-Coupled Receptor Kinase 2

Hepatocellular carcinoma (HCC) is one of the deadliest forms of human liver cancer and does not respond well to conventional therapies. Novel effective treatments are urgently in need. G‐protein‐coupled kinase 2 (GRK2) is unique serine/threonine kinase that involves in many signaling pathways and regulates various essential cellular processes. Altered levels of GRK2 have been linked with several human diseases including cancer. In this study, we investigated a novel approach for HCC treatment by inducing overexpression of GRK2 in human HCC cells. We found that overexpression of GRK2 through recombinant adenovirus transduction inhibits the growth of human HCC cells. BrdU incorporation assay showed that the growth inhibition caused by elevated GRK2 level was due to reduced cell proliferation but not apoptosis. To examine the anti‐proliferative function of increased GRK2 level, we performed cell cycle analysis using propidium iodide staining. We found that the proliferation suppression was associated with G2/M phase cell cycle arrest by the wild‐type GRK2 but not its kinase‐dead K220R mutant. Furthermore, increased levels of wild‐type GRK2 induced upregulation of phosphor‐Ser15 p53 and cyclin B1 in a dose‐dependent manner. Our data indicate that the anti‐proliferative function of elevated GRK2 is associated with delayed cell cycle progression and is GRK2 kinase activity‐dependent. Enforced expression of GRK2 in human HCC by molecular delivery may offer a potential therapeutic approach for the treatment of human liver cancer. J. Cell. Physiol. 227: 2371–2377, 2012.

GRK2 Activity Impairs Cardiac Glucose Uptake and Promotes Insulin Resistance Following Myocardial Ischemia

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the &bgr;-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through &bgr;-adrenergic receptors and metabolism through the insulin receptor.Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile.

Abstract 1234: GRK2 negatively regulates IGF-1R signaling pathway and cyclins in HepG2 cells

G protein-coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of signaling pathways associated with many important cellular processes. Alternation of GRK2 expression has profound effects on cell physiological functions and induces diseases such as heart failure, rheumatoid arthritis, and cancer. In this study, we investigated the role of GRK2 in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentivirus containing GRK2 specific shRNA. Under IGF-1 (50 ng/ml) stimulation, HepG2 cells with reduced GRK2 expression showed increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt. We then treated the cells with gefitinib, an EGFR inhibitor. We found HepG2 cells displayed gefitinib sensitiveness with the increase of drug concentration. Moreover, gefitinib treated cells with reduced expression of GRK2 showed decreased phosphorylation of Akt in a dose dependent manner, but had relatively more IGF-1R expression. However, HepG2 cells with reduced expression of GRK2 did not display any growth difference in culture as compared with the scramble control cells. We further detected that reduced expression of GRK2 induced a small G2/M phase arrest by overexpressing cyclin A, B1, and E. DNA microarray analysis showed decreased transcription levels of IGF binding protein 1 and 4, which were confirmed by quantitative PCR analysis. Our results demonstrated that GRK2 has a role in the regulation of IGF-1R signaling pathway and involves in cell cycle progression in HCC HepG2 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1234. doi:1538-7445.AM2012-1234

G Protein–Coupled Receptor Kinase 2 Activity Impairs Cardiac Glucose Uptake and Promotes Insulin Resistance After Myocardial IschemiaClinical Perspective

Background— Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure. The chronic adrenergic stimulation that accompanies heart failure is a signaling abnormality that leads to the upregulation of G protein–coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part owing to uncoupling of the &bgr;-adrenergic receptor system. In this study, we explored the possibility that enhanced GRK2 expression and activity, as seen during heart failure, can negatively affect cardiac metabolism as part of its pathogenic profile. Methods and Results— Positron emission tomography studies revealed in transgenic mice that cardiac-specific overexpression of GRK2 negatively affected cardiac metabolism by inhibiting glucose uptake and desensitization of insulin signaling, which increases after ischemic injury and precedes heart failure development. Mechanistically, GRK2 interacts with and directly phosphorylates insulin receptor substrate-1 in cardiomyocytes, causing insulin-dependent negative signaling feedback, including inhibition of membrane translocation of the glucose transporter GLUT4. This identifies insulin receptor substrate-1 as a novel nonreceptor target for GRK2 and represents a new pathological mechanism for this kinase in the failing heart. Importantly, inhibition of GRK2 activity prevents postischemic defects in myocardial insulin signaling and improves cardiac metabolism via normalized glucose uptake, which appears to participate in GRK2-targeted prevention of heart failure. Conclusions— Our data provide novel insights into how GRK2 is pathological in the injured heart. Moreover, it appears to be a critical mechanistic link within neurohormonal crosstalk governing cardiac contractile signaling/function through &bgr;-adrenergic receptors and metabolism through the insulin receptor.Background Alterations in cardiac energy metabolism downstream of neurohormonal stimulation play a crucial role in the pathogenesis of heart failure (HF). The chronic adrenergic stimulation that accompanies HF is a signaling abnormality that leads to the up-regulation of G protein-coupled receptor kinase 2 (GRK2), which is pathological in the myocyte during disease progression in part due to uncoupling of the β-adrenergic receptor (βAR) system. In this study we explored the possibility that enhanced GRK2 expression and activity, as seen during HF, can negatively affect cardiac metabolism as part of its pathogenic profile.

Abstract A35: A role for drug resistance in hepatocellular carcinoma: Upregulatation of CD133, activation of IGF-1R signaling, and IGF-1R nuclear translocation

Introduction: Hepatocellular carcinoma (HCC) is an aggressive neoplasm that is resistant to chemotherapy with mechanisms underlying this drug resistance not well understood. CD133 is a known cancer stem cell (CSC) marker whose high expression is associated with aggressive tumor behavior and poor prognosis. The insulin-like growth factor 1 receptor (IGF-1R) signaling pathway has been shown to play a significant role in tumor behavior. To examine possible mechanisms of drug resistance in HCC, we investigated the role of CD133 expression and IGF-1R signaling activation using geftinib. Methods: We evaluated and separated five different human HCC cell lines (Mahlavu, Huh7, HepG2, Hep3B, PLC/PRF/5) using based on CD133 expression. We subjected these cells to geftinib at different doses, and determined IGF-1R signaling pathway activation using immunoblotting against relevant proteins in the pathway. CD133 mRNA level was examined using quantitative real-time PCR and its expression level was detected using immunofluorescence imaging and flow cytometry after drug treatment. Cell viability and sensitivity to drug treatment were measured using the MTT assay. Results: HCC cell lines showed variable percentages of total cell populations expressing CD133: Mahlavu 0.12 %, HepG2 46 %, Huh7 80.2 %, Hep3B 98.2 %, and PLC/PRF/5 83.5 %. Mahlavu and PLC/PRF/5 were found to be the most resistant cell lines even at higher doses (17.5 uM). Since Mahlavu cells displayed reduced CD133 expression, experiments were focused on this cell line to evaluate the CD133 expression changes after geftinib treatment. After three rounds (9 days) of high dose geftinib treatment, CD133 expression was increased in a dose dependent manner; the percentages of total cell populations expressing CD133 increased to 43.9 % at 10 uM concentration and 52.9 % at 15 uM concentration as compared to 0.12 % in untreated cells. In addition, we found a significant increase in IGF-1R and AKT phosphorylation as well as IGFBP3 expression in those geftinib treated cells. Furthermore, after cell sorting of Huh7 cells by CD133 expression using flow cytometry, we observed that CD133 positive cells showed more resistant to geftinib treatment. Immunofluorescence imaging analysis demonstrated that increased IGF-1R protein translocated to nuclei after geftinib treatment. Conclusion: Geftinib treatment enhanced CD133 expression level and activated IGF-1R signaling in HCC Mahlavu cells, one of the possible mechanism could be the nuclear translocation of IGF-1R. Our findings underscore the importance of a combined therapeutic approach with geftinib and IGF-1R blockage in the treatment of human HCC. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A35.