Keng-Li Lan

A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family α subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Gα subunit Gpa1sst that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780–5784), site-directed mutagenesis of αo and αi1 was done. G184S αo and G183S αi1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G → S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nm, respectively, for aluminum fluoride-activated wild type αo and αi1 to compete with fluorescein isothiocyanate-αo binding to glutathioneS-transferase-RGS4. The G → S mutant proteins showed a greater than 30–100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in αo and αi1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation Gα SPECIFICITY OF RGS4 AND RGS7

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits speeding deactivation. Gα deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Gα accompanying GTP hydrolysis and Gα deactivation on the millisecond time scale. Basal k cat values for Gαo, Gαi1, and Gαi2 at 20 °C were similar (0.025–0.033 s−1). GlutathioneS-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305–453) enhanced the rate of Gα deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s−1 at 3 μm, giving a catalytic efficiency of 1.7–2.8 × 106 m −1 s−1 for all three Gα subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 × 106 m −1s−1 toward Gαo, Gαi2, and Gαi1, respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Gα subunits tested, but it is specific for Gαo over Gαi1 or Gαi2. Furthermore, the specificity of RGS7 for Gαo does not depend on N- or C-terminal extensions or a Gβ5 subunit but resides in the RGS domain itself.

Strategies to target HER2/neu overexpression for cancer therapy

Amplification or overexpression of the HER2/neu (also known as erbB-2) gene has been noted in various types of human cancers. In addition to malignant transformation, the activation of signaling pathways of HER2/neu enhances various metastasis-associated properties and may render cancer cells resistant to conventional therapies. This, at least partially, contributes to the poor prognosis and lower survival rate of patients. Many studies have demonstrated that repression of HER2/neu overexpression suppresses the malignant phenotypes of cancer cells. Therefore, various novel HER2/neu-blocking agents have been developed, several of which have been tested in clinical trials with satisfactory results, including trastuzumab, a HER2/neu monoclonal antibody that has been approved by the FDA in the treatment of HER2/neu-overexpressing breast cancer patients. In this article, we intend to discuss the biological relevance and significance of HER2/neu overexpression in tumorigenesis, metastasis, and resistance to conventional therapy. We also summarize the currently available strategies and combination therapies targeting HER2/neu-overexpressing cancer cells. Although the optimal treatment for HER2/neu-overexpressing cancer patients remains elusive, the initial success of trastuzumab indicates that HER2/neu is a good target for cancer therapy. Further elucidation of HER2/neu-mediated pathways and downstream molecules is critical to provide alternative therapies, overcome drug resistance, and improve the therapeutic outcome for HER2/neu-overexpressing cancer patients.

RGS-Insensitive G-Protein Mutations to Study the Role of Endogenous RGS Proteins

Regulator of G-protein signaling (RGS) proteins are very active GTPase-accelerating proteins (GAPs) in vitro and are expected to reduce signaling by G-protein coupled receptors in vivo. A novel method is presented to assess the in vivo role of RGS proteins in the function of a G protein in which Galpha subunits do not bind to RGS proteins or respond with enhanced GTPase activity. A point mutation in the switch I region of Galpha subunits (G184S Galpha(o) and G183S Galpha(i1)) blocks the interaction with RGS proteins but leaves intact the ability of Galpha to couple to betagamma subunits, receptors, and downstream effectors. Expression of the RGS-insensitive mutant G184S Galpha(o) in C6 glioma cells with the micro-opioid receptor dramatically enhances adenylylcyclase inhibition and activation of extracellular regulated kinase. Introducing the same G184S Galpha(o) protein into embryonic stem (ES) cells by gene targeting allows us to assess the functional importance of the endogenous RGS proteins using in vitro differentiation models and in intact mice. Using ES cell-derived cardiocytes, spontaneous and isoproterenol-stimulated beating rates were not different between wild-type and G184S Galpha(o) mutant cells; however, the bradycardiac response to adenosine A1 receptor agonists was enhanced significantly (seven-fold decrease EC50) in Galpha(o)RGSi mutant cells compared to wild-type Galpha(o), indicating a significant role of endogenous RGS proteins in cardiac automaticity regulation. The approach of using RGS-insensitive Galpha subunit knockins will reveal the role of RGS protein-mediated GAP activity in signaling by a given G(i/o) protein. This will reveal the full extent of RGS regulation and will not be confounded by redundancy in the function of multiple RGS proteins.

Multiple effects of honokiol on the life cycle of hepatitis C virus

Honokiol, a small active molecular compound extracted from magnolia, has recently been shown to inhibit hepatitis C virus (HCV) infection in vitro.

Biodistribution and pharmacokinetics of 188Re-liposomes and their comparative therapeutic efficacy with 5-fluorouracil in C26 colonic peritoneal carcinomatosis mice.

Background Nanoliposomes are designed as carriers capable of packaging drugs through passive targeting tumor sites by enhanced permeability and retention (EPR) effects. In the present study the biodistribution, pharmacokinetics, micro single-photon emission computed tomography (micro-SPECT/CT) image, dosimetry, and therapeutic efficacy of 188Re-labeled nanoliposomes (188Re-liposomes) in a C26 colonic peritoneal carcinomatosis mouse model were evaluated. Methods Colon carcinoma peritoneal metastatic BALB/c mice were intravenously administered 188Re-liposomes. Biodistribution and micro-SPECT/CT imaging were performed to determine the drug profile and targeting efficiency of 188Re-liposomes. Pharmacokinetics study was described by a noncompartmental model. The OLINDA|EXM® computer program was used for the dosimetry evaluation. For therapeutic efficacy, the survival, tumor, and ascites inhibition of mice after treatment with 188Re-liposomes and 5-fluorouracil (5-FU), respectively, were evaluated and compared. Results In biodistribution, the highest uptake of 188Re-liposomes in tumor tissues (7.91% ± 2.02% of the injected dose per gram of tissue [%ID/g]) and a high tumor to muscle ratio (25.8 ± 6.1) were observed at 24 hours after intravenous administration. The pharmacokinetics of 188Re-liposomes showed high circulation time and high bioavailability (mean residence time [MRT] = 19.2 hours, area under the curve [AUC] = 820.4%ID/g*h). Micro-SPECT/CT imaging of 188Re-liposomes showed a high uptake and targeting in ascites, liver, spleen, and tumor. The results were correlated with images from autoradiography and biodistribution data. Dosimetry study revealed that the 188Re-liposomes did not cause high absorbed doses in normal tissue but did in small tumors. Radiotherapeutics with 188Re-liposomes provided better survival time (increased by 34.6% of life span; P < 0.05), tumor and ascites inhibition (decreased by 63.4% and 83.3% at 7 days after treatment; P < 0.05) in mice compared with chemotherapeutics of 5-fluorouracil (5-FU). Conclusion The use of 188Re-liposomes for passively targeted tumor therapy had greater therapeutic effect than the currently clinically applied chemotherapeutics drug 5-FU in a colonic peritoneal carcinomatosis mouse model. This result suggests that 188Re-liposomes have potential benefit and are safe in treating peritoneal carcinomatasis of colon cancer.

Decoy receptor 3 enhances tumor progression via induction of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.

Honokiol inhibits hypoxia-inducible factor-1 pathway.

Purpose: Hypoxia-inducible factor-1α (HIF-1α) plays a pivotal role in the reaction of a tumour to hypoxia. In this study, we examined the inhibitory effect of a natural compound, honokiol, on HIF-1α activity and tumour growth in combination with radiation. Methods: The inhibitory effect of honokiol on hypoxia-responsive element (HRE) controlled luciferase activity and HIF-1α accumulations stimulated by CoCl2, or hypoxia was examined. Effect of honokiol on HIF-1α levels within hypoxic tumour microenvironment was investigated by immunohistochemical and in vivo bioluminescent studies. The in vivo radiosensitising activity of honokiol was evaluated with subcutaneous murine colon carcinoma, CT26, xenografts of BALB/c mice treated with honokiol, radiation, or both. Results: Suppression of luciferase (luc) activity in HRE-luc stable cells by honokiol was in agreement with the results of decreased HIF-1α accumulation. In CT26-HRE-luc tumour-bearing mice, the inhibitory effect of intraperitoneally injected honokiol on HIF-1α-regulated luciferase activities induced by either CoCl2 or radiation could be monitored non-invasively. Lastly, honokiol in combination with irradiation produced synergistic delay of CT26 tumour growth. Conclusions: Our data suggest that honokiol can exert its anticancer activity as a HIF-1α inhibitor by reducing HIF-1α protein level and suppressing the hypoxia-related signaling pathway. The animal experiment indicates that honokiol improves the therapeutic efficacy of radiation.

Endostatin-Cytosine Deaminase Fusion Protein Suppresses Tumor Growth by Targeting Neovascular Endothelial Cells

Endostatin, an angiogenesis inhibitor tested in multiple clinical trials, selectively targets neovascular endothelial cells, suppressing tumor growth. To enhance the therapeutic efficacy of endostatin, we fused endostatin with cytosine deaminase, which converts a prodrug 5-flucytosine into a cytotoxic 5-fluorouracil. This therapeutic strategy was developed based on the observation that the endostatin-green fluorescence protein gene and endostatin-luciferase gene selectively target to endothelial cells in vitro and to the tumor site in vivo, respectively. When we used the endostatin-cytosine deaminase fusion protein to treat s.c. grafted tumors or experimental metastasis tumors, our results showed that endostatin-cytosine deaminase treatment provided stronger tumor growth suppression and increased mean survival time of the mice compared with the treatments of endostatin alone, cytosine deaminase alone, or endostatin plus cytosine deaminase. The endostatin-cytosine deaminase protein significantly inhibited the growth of endothelial cells and preferentially induced tumor cell apoptosis. This endostatin-cytosine deaminase fusion approach opens an avenue for cancer-targeting therapy.

A DNA vaccine against cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) prevents tumor growth

Co-stimulatory signaling pathway triggered by the binding of B7.1/B7.2 (CD80/86) of antigen-presenting cells (APCs) to CD28 of T cells is required for optimal T-cell activation. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Ipilimumab, a monoclonal antibody against CTLA-4, has shown positive efficacy in a pivotal clinical trial for the treatment of metastatic melanoma and was approved by FDA. However, the cost of monoclonal antibody-based therapeutics might limit the number of patients treated. To develop a novel therapeutics specifically targeting CTLA-4, we constructed a DNA vaccine by cloning the sequence of CTLA-4 fused with a transmembrane domain sequence of placental alkaline phosphatase (PLAP) into a mammalian expression plasmid, pVAC-1. Immunization with the resulting construct, pVAC-1-hCTLA-4, elicited antibody specific to human CTLA-4 with cross reactivity to murine CTLA-4, which was sufficient for inhibiting B16F10 tumor growth in c57BL/6 mice in the absence of measurable toxicity. Coupling liposome with pVAC-1-mCTLA-4 could break tolerance to self-antigen in BALB/c mice and induce potent immunity against murine CTLA-4, and suppress growth of subcutaneous renal cell carcinoma (Renca).