Shuyi Si

Berberine reduces insulin resistance through protein kinase C–dependent up-regulation of insulin receptor expression

Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

MicroRNAs 185, 96, and 223 Repress Selective High-Density Lipoprotein Cholesterol Uptake through Posttranscriptional Inhibition

ABSTRACT Hepatic scavenger receptor class B type I (SR-BI) plays an important role in selective high-density lipoprotein cholesterol (HDL-C) uptake, which is a pivotal step of reverse cholesterol transport. In this study, the potential involvement of microRNAs (miRNAs) in posttranscriptional regulation of hepatic SR-BI and selective HDL-C uptake was investigated. The level of SR-BI expression was repressed by miRNA 185 (miR-185), miR-96, and miR-223, while the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-HDL was decreased by 31.9% (P < 0.001), 23.9% (P < 0.05), and 15.4% (P < 0.05), respectively, in HepG2 cells. The inhibition of these miRNAs by their anti-miRNAs had opposite effects in these hepatic cells. The critical effect of miR-185 was further validated by the loss of regulation in constructs with mutated miR-185 target sites. In addition, these miRNAs directly targeted the 3′ untranslated region (UTR) of SR-BI with a coordinated effect. Interestingly, the decrease of miR-96 and miR-185 coincided with the increase of SR-BI in the livers of ApoE KO mice on a high-fat diet. These data suggest that miR-185, miR-96, and miR-223 may repress selective HDL-C uptake through the inhibition of SR-BI in human hepatic cells, implying a novel mode of regulation of hepatic SR-BI and an important role of miRNAs in modulating cholesterol metabolism.

DH334, a β-carboline anti-cancer drug, Inhibits the CDK activity of budding yeast

The β-carboline alkaloids present in medicinal plants, such as Peganum harmala and Eurycoma longifolia, have recently drawn attention due to their antitumor activities. Further mechanistic studies indicate that β-carboline derivatives inhibit DNA topoisomerases and interfere with DNA synthesis. Moreover, some β-carboline compounds are specific inhibitors of cyclin dependent kinases (CDKs). In this study we used budding yeast as a model system to investigate the antitumor mechanism of β-carboline drugs. We found that DH334, a β-carboline derivative, inhibits the growth of budding yeast. Strikingly, deletion of SIC1, which encodes the budding yeast CDK inhibitor, results in resistance to DH334. In contrast, yeast cells defective for Sic1 degradation exhibit more pronounced sensitivity to DH334. The presence of DH334 causes accumulation of yeast cells in G1 phase, indicating that DH334 blocks cell cycle initiation. We further demonstrated that DH334 inhibits CDK activity as indicated by the decreased phosphorylation of a CDK substrate. All these data suggest that the inhibition of CDK contributes to the toxicity of β-carboline derivatives to budding yeast. DH334 also inhibits the kinase activity of Cdk2/CyclinA in vitro. Therefore, we speculate that the antitumor activity of β-carboline drugs could be attributable to their inhibition of CDK.

Substituted Benzothiophene or Benzofuran Derivatives as a Novel Class of Bone Morphogenetic Protein-2 Up-Regulators: Synthesis, Structure−Activity Relationships, and Preventive Bone Loss Efficacies in Senescence Accelerated Mice (SAMP6) and Ovariectomized Rats

In this work, substituted benzothiophene and benzofuran compounds were found to be a new class of potential anabolic agents by enhancing BMP-2 expression. A series of benzothiophene and benzofuran derivatives have been synthesized, and their activities of up-regulating BMP-2 and bone loss prevention efficacies in SAMP6 mice and OVX rats have been studied. Benzothiophenes 1, 3, 14, 4a, 7a, 8a, and benzofuran analogue 2 showed higher BMP-2 up-regulation rates in vitro. Compound 8a was found to significantly affect the bone formation rate of tested SAMP6 mice. Compound 1 showed an improved bone quality in SAMP6 mice and also showed activity in OVX rats. We have demonstrated that substituted benzothiophene and benzofuran derivatives, especially compounds 1 and 8a, enhance BMP-2 expression in vitro and in vivo and stimulate bone formation and trabecular connectivity restoration in vivo. The compounds represent potential leads in the development of a new class of anabolic agents.

Jadomycin B, an Aurora-B kinase inhibitor discovered through virtual screening

Aurora kinases have emerged as promising targets for cancer therapy because of their critical role in mitosis. These kinases are well-conserved in all eukaryotes, and IPL1 gene encodes the single Aurora kinase in budding yeast. In a virtual screening attempt, 22 compounds were identified from nearly 15,000 microbial natural products as potential small-molecular inhibitors of human Aurora-B kinase. One compound, Jadomycin B, inhibits the growth of ipl1-321 temperature-sensitive mutant more dramatically than wild-type yeast cells, raising the possibility that this compound is an Aurora kinase inhibitor. Further in vitro biochemical assay using purified recombinant human Aurora-B kinase shows that Jadomycin B inhibits Aurora-B activity in a dose-dependent manner. Our results also indicate that Jadomycin B competes with ATP for the kinase domain, which is consistent with our docking prediction. Like other Aurora kinase inhibitors, Jadomycin B blocks the phosphorylation of histone H3 on Ser10 in vivo. We also present evidence suggesting that Jadomycin B induces apoptosis in tumor cells without obvious effects on cell cycle. All the results indicate that Jadomycin B is a new Aurora-B kinase inhibitor worthy of further investigation. [Mol Cancer Ther 2008;7(7):2386–93]

DH166, a beta-carboline derivative, inhibits the kinase activity of PLK1

A better way to treat complex diseases such as cancer is to aim for several targets at once. Beta-carboline derivatives have been shown to have anticancer activity, but these compounds may target several enzymes required for cell division. Polo-like kinases (PLKs) are well conserved serine/threonine kinases and PLK1 plays multiple roles in cell proliferation. Thus, PLK1 is one of the attractive mitotic targets for anticancer drugs. We found that DH166, a beta-carboline derivative, inhibits the growth of cdc5-2 temperature-sensitive mutant more profoundly than wild-type yeast cells. Because Cdc5 is the human PLK1 homologue in budding yeast, this observation indicates that DH166 might be a PLK1 inhibitor. Indeed, DH166 inhibits the kinase activity of purified PLK1 at low micromolar concentration in an ATP-competitive manner, which is consistent with the docking result based on the crystal structure of PLK1. In addition, DH166 blocks cancer cell proliferation, causes a mitotic arrest, increases cyclin B1 accumulation, induces aberrant mitotic spindles and apoptosis, presumably due to the down-regulation of PLK1. Although beta-carboline derivatives have been demonstrated to show antitumor activities through multiple mechanisms, our data indicate for the first time that their cytotoxicity to tumor cells might be attributable to the inhibition of PLK1 as well.

Identification of Upregulators of Human ATP-Binding Cassette Transporter A1 via High-Throughput Screening of a Synthetic and Natural Compound Library

The ATP-binding cassette transporter A1 (ABCA1) is a membrane transporter that directly contributes to high-density lipoprotein (HDL) biogenesis by mediating the cellular efflux of cholesterol and phospholipids to lipid-poor apolipoprotein A-I. Therefore, identification of a novel upregulator of ABCA1 would be beneficial for atherosclerosis prevention and/or therapy because of its pivotal role in cholesterol homeostasis and HDL metabolism. In this study, a high-throughput assay method for ABCA1 upregulators was developed and used for screening a synthetic and natural compound library. The cell-based high-throughput screen is conducted in a 96-well format using the human hepatoma HepG2 cells stably transfected with ABCA1 promoter-luciferase construct and calibrated with reference ABCA1 upregulators (oxysterols, 9-cis-retinoic acid, thiazolidinediones, cyclic adenosine monophosphate, verapamil, fenofibrate, and oncostatin M). Among 2600 compounds, 4 microbial compounds (pyrromycin, aclarubicin, daidzein, and pratensein) were picked up as hits by the high-throughput screening assay, and those compounds were further identified as upregulators of ABCA1 expression by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. (Journal of Biomolecular Screening 2008:648-656)

Identification of Upregulators of BMP2 Expression via High-Throughput Screening of a Synthetic and Natural Compound Library

Bone morphogenetic protein II (BMP2), a member of the transforming growth factor—β (TGF-β) superfamily, is highly expressed in osteoblasts and is a crucial regulator of osteogenic differentiation. Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis, and it may be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. To discover new agents that enhance the expression of the mouse BMP2, the authors developed a high-throughput assay to screen a synthetic and natural compound library. The cell-based high-throughput screen was conducted in 96-well plates using the clonal murine calvarial MC3T3-E1 cells. These cells were stably transfected with mouse BMP2 promoter-luciferase and calibrated with the known antiosteoporosis compound genistein. Among 3192 compounds screened, 3 agents (daidzein, formononetin, and 2-Acetyldibenzothiophene) were picked up by the high-throughput screening assay, and those compounds were identified as upregulators of BMP2 expression by real-time quantitative reverse transcription—polymerase chain reaction and flow cytometry. Thus, it is demonstrated that this screening model is useful for identifying lead compounds to treat osteoporosis and maintain bone metabolism balance.

Identification of trichostatin A as a novel transcriptional up-regulator of scavenger receptor BI both in HepG2 and RAW 264.7 cells

Scavenger receptor class B type I (SR-BI) and its human homologue CLA-1 plays an important role in reverse cholesterol transport (RCT). Using a previously established cell-based CLA-1 up-regulator screening assay, one of the positive strains, 04-9179, presented potent activity in elevating CLA-1 transcriptional level. We report here the identification of an active compound 9179A as a known compound trichostatin A (TSA), and its effects on CLA-1/SR-BI expression both in HepG2 human hepatoma cells and RAW 264.7 murine macrophage cells in vitro. The results showed that the mRNA and protein level of CLA-1/SR-BI were significantly up-regulated by 9179A both in HepG2 and RAW 264.7 cells. Corresponding to this, the uptake of DiI-HDL by both cells and the efflux of [(3)H]cholesterol by RAW 264.7 cells were increased by 9179A in dose-dependent manner. ABCA1 was also increased but SR-A decreased by 9179A in RAW 264.7 cells. Using a combination of reporter assays with various deletion in CLA-1 promoter and electrophoretic mobility shift assay, we demonstrated that -419/-232 bp fragment of the CLA-1 promoter mediated the effects of 9179A (i.e., TSA). Together, these studies identified TSA as a novel up-regulator of CLA-1/SR-BI both in HepG2 and RAW 264.7 cells.

A series of beta-carboline derivatives inhibit the kinase activity of PLKs.

Polo-like kinases play an essential role in the ordered execution of mitotic events and 4 mammalian PLK family members have been identified. Accumulating evidence indicates that PLK1 is an attractive target for anticancer drugs. In this paper, a series of beta-carboline derivatives were synthesized and three compounds, DH281, DH285 and DH287, were identified as potent new PLK inhibitors. We employed various biochemical and cellular approaches to determine the effects of these compounds on the activity of PLK1 and other mitotic kinases and on cell cycle progression. We found that these three compounds could selectively inhibit the kinase activity of purified PLK1, PLK2 and PLK3 in vitro. They show strong antitumor activity against a number of cancer cell lines with relatively low micromolar IC50s, but are relatively less toxic to non-cancer cells (MRC5). Moreover, these compounds could induce obvious accumulation of HeLa cells in G2/M and S phases and trigger apoptosis. Although MRC5 cells show clear S-phase arrest after treatment with these compounds, the G2/M arrest and apoptosis are less insignificant, indicating the distinct sensitivity between normal and cancer cells. We also found that HeLa cells treated with these drugs exhibit monopolar spindles and increased Wee1 protein levels, the characteristics of cells treated with PLK1 inhibitors. Together, these results demonstrate that DH281, DH285 and DH287 beta-carboline compounds are new PLK inhibitors with potential for cancer treatment.