Fangfang Lai

Chlorogenic acid inhibits glioblastoma growth through repolarizating macrophage from M2 to M1 phenotype

Glioblastoma is an aggressive tumor that is associated with distinctive infiltrating microglia/macrophages populations. Previous studies demonstrated that chlorogenic acid (5-caffeoylquinic acid, CHA), a phenolic compound with low molecular weight, has an anti-tumor effect in multiple malignant tumors. In the present study, we focused on the macrophage polarization to investigate the molecular mechanisms behind the anti-glioma response of CHA in vitro and in vivo. We found that CHA treatment increased the expression of M1 markers induced by LPS/IFNγ, including iNOS, MHC II (I-A/I-E subregions) and CD11c, and reduced the expression of M2 markers Arg and CD206 induced by IL-4, resulting in promoting the production of apoptotic-like cancer cells and inhibiting the growth of tumor cells by co-culture experiments. The activations of STAT1 and STAT6, which are two crucial signaling events in M1 and M2-polarization, were significantly promoted and suppressed by CHA in macrophages, respectively. Furthermore, In G422 xenograft mice, CHA increased the proportion of CD11c-positive M1 macrophages and decreased the distribution of CD206-positive M2 macrophages in tumor tissue, consistent with the reduction of tumor weight observed in CHA-treated mice. Overall these findings indicated CHA as a potential therapeutic approach to reduce glioma growth through promoting M1-polarized macrophage and inhibiting M2 phenotypic macrophage.

Rutaecarpine suppresses atherosclerosis in ApoE-/- mice through upregulating ABCA1 and SR-BI within RCT.

ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE−/−) mice treated with RUT for 8 weeks showed ∼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE−/− mice. Meanwhile, RUT treatment significantly increased the fecal 3H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE−/− mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.

LXY6090 – a novel manassantin A derivative – limits breast cancer growth through hypoxia-inducible factor-1 inhibition

Hypoxia-inducible factor-1 (HIF-1) represents a novel antitumor target owing to its involvement in vital processes considered hallmarks of cancer phenotypes. Manassantin A (MA) derived from Saururus cernuus has been reported as a selective HIF-1 inhibitor. Herein, the structure of MA was optimized to achieve new derivatives with simple chemical properties while retaining its activity. LXY6090 was designed to replace the central tetrahydrofuran moiety of MA with a cyclopentane ring and was identified as a potent HIF-1 inhibitor with an IC50 value of 4.11 nM. It not only inhibited the activity of HIF-1 in breast cancer cells but also downregulated the protein level of HIF-1α, which depended on von Hippel–Lindau for proteasome degradation. The related biological evaluation showed that the activity of HIF-1 target genes, VEGF and IGF-2, was decreased by LXY6090 in breast cancer cell lines. LXY6090 presented potent antitumor activity in vitro. Furthermore, LXY6090 showed in vivo anticancer efficacy by decreasing the HIF-1α expression in nude mice bearing MX-1 tumor xenografts. In conclusion, our data provide a basis for the future development of the novel compound LXY6090 as a potential therapeutic agent for breast cancer.

Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPARγ-LXRα-ABCA1 pathway.

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50=0.09 μM). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ; EC50=5.2-9.3 μM). Liver X receptor α (LXRα) is a target gene of PPARγ and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXRα protein expression in HepG2 cells. Addition of PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPARγ. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPARγ-LXRα-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

A Morphological identification cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead cells

Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds.

LB-1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF-1α and Stat3 Signaling.

Hypoxia is widely present in pancreatic cancer and subsequently causes the overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) and signal transducer and activator of transcription‐3 (Stat3). HIF‐1α and Stat3 function cooperatively to regulate a number of downstream genes that are implicated in tumorigenesis. Thus, inhibition of HIF‐1α and Stat3 is a potential therapeutic strategy for pancreatic cancer. In this study, we explored how LB‐1, a novel triptolide (LA) derivative, exerted its antitumor effect through blockade of HIF‐1α and Stat3 signaling. Our data showed that LB‐1 was able to inhibit the proliferation and colony formation of Mia‐PaCa2 and SW1990 cells. LB‐1 suppressed HIF‐1α protein accumulation by promoting its proteasome degradation and reducing transactivation. Moreover, the silence of HIF‐1α by shRNA partially prevented the proliferation inhibition triggered by LB‐1. As expected, LB‐1 also decreased Stat3 protein accumulation and blocked the physical interactions between HIF‐1α/p300/phosphor‐Stat3 (p‐Stat3) at the pharmacological concentration to reduce VEGF expression, thereby hypoxia‐induced angiogenesis. In the Mia‐PaCa2 nude xenograft model, therapeutic treatment with LB‐1 significantly inhibited tumor growth and had minimal systemic toxicity compared to the mother drug LA. Furthermore, in accordance with in vitro results, HIF‐1α activation and Stat3 expression in tumors were blocked by LB‐1 through mTOR‐dependent pathway. Taken together, these results illustrate that, as a potent inhibitor of HIF‐1α and Stat3 signaling, LB‐1 exhibits antitumor effect and could be potentially used to treat pancreatic cancer. J. Cell. Physiol. 230: 2212–2223, 2015.

CAT 3 , a prodrug of 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro[9,10-b]-indolizidine, circumvents temozolomide-resistant glioblastoma via the Hedgehog signaling pathway, independently of O 6 -methylguanine DNA methyltransferase expression

Purpose Glioblastoma multiforme (GBM) is a malignant high-grade glioma with a poor clinical outcome. Temozolomide (TMZ) is the first-line GBM chemotherapy; however, patients commonly develop resistance to its effects. Materials and methods We investigated the antitumor activity of CAT3 in TMZ-resistant glioblastoma cell lines U251/TMZ and T98G. Orthotopic and subcutaneous mice tumor models were used to investigate the effects of various treatment regimes. Results We found that PF403, the active metabolite of CAT3, inhibited proliferation of both cell lines. PF403 repressed the Hedgehog signaling pathway in the U251/TMZ cell line, reduced O6-methylguanine DNA methyltransferase (MGMT) expression, and abolished the effects of the Shh pathway. Moreover, PF403 blocked the Hedgehog signaling pathway in T98G MGMT-expressing cells and downregulated the expression of MGMT. CAT3 suppressed growth in the U251/TMZ orthotopic and T98G subcutaneous xenograft tumor models in vivo. We also demonstrated that inhibition of the Hedgehog pathway by PF403 counteracted TMZ resistance and enhanced the antitumor activity of TMZ in vitro and in vivo. Conclusion These results indicate that CAT3 is a potential therapeutic agent for TMZ-resistant GBM.

Discovery of novel quinazoline-2,4(1 H ,3 H )-dione derivatives as potent PARP-2 selective inhibitors

The PARP-2 selective inhibitor is important for clarifying specific roles of PARP-2 in the pathophysiological process and developing desired drugs with reduced off-target side effects. In this work, a series of novel quinazoline-2,4(1H,3H)-dione derivatives was designed and synthesized to explore isoform selective PARP inhibitors. As a result, compound 11a (PARP-1 IC50=467nM, PARP-2 IC50=11.5nM, selectivity PARP-1/PARP-2=40.6) was disclosed as the most selective PARP-2 inhibitor with high potency to date. The binding features of compound 11a within PARP-1 and PARP-2 were investigated respectively to provide useful insights for the further construction of new isoform selective inhibitors of PARP-1 and PARP-2 by using CDOCKER program.

The Development of a Biotinylated NAD+-Applied Human Poly(ADP-Ribose) Polymerase 3 (PARP3) Enzymatic Assay:

Poly(ADP-ribose) polymerase 3 (PARP3) is an important member of the PARP family and shares high structural similarities with both PARP1 and PARP2. The biological roles of PARP3 are currently under investigation; however, several key reports indicate the integral roles of PARP3 in DNA damage repair, and thus it has been investigated as a novel target in oncology. It is clear that the identification of selective PARP3 inhibitors would further advance the understanding of the biological roles of PARP3. Herein, we describe a modified PARP3 screening assay using biotinylated NAD+ as the specialized substrate. This method relies on the activity of PARP3 to transfer the biotinylated NAD+ onto a histone protein to form ADP-ribosylated histone. The biotin label on this histone protein is then detected and quantifies the intrinsic enzymatic activity of PARP3. We optimized the assay with respect to the histone, NAD+/biotinylated NAD+ mixture, DNA, and PARP3. Our developed screening system was then validated with a reported selective PARP3 inhibitor, ME0328, as well as utilizing five other clinically available PARP1/2 inhibitors. We demonstrated that our assay system was sensitive, efficient, and economical, and we reason that it could be useful for the development of highly selective PARP3 inhibitors in the future.

Quantitative Evaluation of in Vivo Target Efficacy of Anti-tumor Agents via an Immunofluorescence and EdU Labeling Strategy

Current methods used to evaluate in vivo target efficacy of selected compound include western blot to semi-quantitatively analyze protein expression. However, problems arise as it is difficult to compare in vivo target efficacy of anti-tumor agents with the same mode of action. It is therefore desirable to develop a protocol that can quantitatively display in vivo target efficacy while also providing other useful information. In this study EdU labeling was used to mark out the proliferating area. The tumor tissue was accordingly divided into proliferating and non-proliferating areas. Fifteen tumor related proteins were stained by immunofluorescence and were found to express in either the proliferating or non-proliferating areas. This allows the quantitative analysis of protein expressions within the precise area. With simple image analysis, our method gave precise percent changes of protein expression and cell proliferation between the drugs treated group and the control group. Additional information, such as, the status of protein expression can also be obtained. This method exhibits high sensitivity, and provides a quantitative approach for in vivo evaluation of target efficacy.