Pharmacological and mechanistic study of PS1, a Pdia4 inhibitor, in ß-cell pathogenesis and diabetes in db/db mice.

Pdia4 has been characterized as a key protein that positively regulates ß-cell failure and diabetes via ROS regulation. Here, we investigated the function and mechanism of PS1, a Pdia4 inhibitor, in ß-cells and diabetes. We found that PS1 had an IC50 of 4 µM for Pdia4. Furthermore, PS1 alone and in combination with metformin significantly reversed diabetes in db/db mice, 6 to 7 mice per group, as evidenced by blood glucose, glycosylated hemoglobin A1c (HbA1c), glucose tolerance test, diabetic incidence, survival and longevity (P < 0.05 or less). Accordingly, PS1 reduced cell death and dysfunction in the pancreatic ß-islets of db/db mice as exemplified by serum insulin, serum c-peptide, reactive oxygen species (ROS), islet atrophy, and homeostatic model assessment (HOMA) indices (P < 0.05 or less). Moreover, PS1 decreased cell death in the ß-islets of db/db mice. Mechanistic studies showed that PS1 significantly increased cell survival and insulin secretion in Min6 cells in response to high glucose (P < 0.05 or less). This increase could be attributed to a reduction in ROS production and the activity of electron transport chain complex 1 (ETC C1) and Nox in Min6 cells by PS1. Further, we found that PS1 inhibited the enzymatic activity of Pdia4 and mitigated the interaction between Pdia4 and Ndufs3 or p22 in Min6 cells (P < 0.01 or less). Taken together, this work demonstrates that PS1 negatively regulated ß-cell pathogenesis and diabetes via reduction of ROS production involving the Pdia4/Ndufs3 and Pdia4/p22 cascades.

Synthesis and biological evaluation of C-4 substituted phenoxazine-bearing hydroxamic acids with potent class II histone deacetylase inhibitory activities.

Class II histone deacetylases (HDACs) are considered as potential targets to treat Alzheimer's disease (AD). Previously, C-3 substituted phenothiazine-containing compounds with class II HDAC-inhibiting activities was found to promote neurite outgrowth. This study replaced phenothiazine moiety with phenoxazine that contains many C-3 and C-4 substituents. Some resulting compounds bearing the C-4 substituent on a phenoxazine ring displayed potent class II HDAC inhibitory activities. Structure-activity relationship (SAR) of these compounds that inhibited HDAC isoenzymes was disclosed. Molecular modelling analysis demonstrates that the potent activities of C-4 substituted compounds probably arise from π-π stacked interactions between these compounds and class IIa HDAC enzymes. One of these, compound 7d exhibited the most potent class II HDAC inhibition (IC50= 3-870 nM). Notably, it protected neuron cells from H2O2-induced neuron damage at sub-µM concentrations, but with no significant cytotoxicity. These findings show that compound 7d is a lead compound for further development of anti-neurodegenerative agents.

Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells.

The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.

Identification of a dual FLT3 and MNK2 inhibitor for acute myeloid leukemia treatment using a structure-based virtual screening approach.

Fms-like tyrosine kinase 3 (FLT3) is considered a promising therapeutic target for acute myeloid leukemia (AML) in the clinical. However, monotherapy with FLT3 inhibitor is usually accompanied by drug resistance. Dual inhibitors might be therapeutically beneficial to patients with AML due to their ability to overcome drug resistance. Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) phosphorylate eukaryotic translation initiation factor 4E (eIF4E), which brings together the RAS/RAF/ERK and PI3K/AKT/mTOR oncogenic pathways. Therefore, dual inhibition of FLT3 and MNK2 might have an additive effect against AML. Herein, a structure-based virtual screening approach was performed to identify dual inhibitors of FLT3 and MNK2 from the ChemDiv database. Compound K783-0308 was identified as a dual inhibitor of FLT3 and MNK2 with IC50 values of 680 and 406 nM, respectively. In addition, the compound showed selectivity for both FLT3 and MNK2 in a panel of 82 kinases. The structure-activity relationship analysis and common interactions revealed interactions between K783-0308 analogs and FLT3 and MNK2. Furthermore, K783-0308 inhibited MV-4-11 and MOLM-13 AML cell growth and induced G0/G1 cell cycle arrest. Taken together, the dual inhibitor K783-0308 showed promising results and can be potentially optimized as a lead compound for AML treatment.

1,2,3,4,6-Penta-O-galloyl-d-glucose Interrupts the Early Adipocyte Lifecycle and Attenuates Adiposity and Hepatic Steatosis in Mice with Diet-Induced Obesity.

Phytochemicals that interrupt adipocyte lifecycle can provide anti-obesity effects. 1,2,3,4,6-penta-O-galloyl-d-glucose (PGG) is a tannin with two isomers that occurs widely in plants and exhibits various pharmacological activities. The aim of the investigation is to comprehensively examine effects of PGG isomer(s) on adipocyte lifecycle and diet-induced obesity. Human mesenchymal stem cells (hMSC), 3T3-L1 fibroblasts, and H4IIE hepatoma cells were used to determine the effects of PGG isomers on cell viability and adipogenesis. Mice with diet-induced obesity were generated from male C57/BL6 mice fed with a 45% high fat diet. Oral administration of ß-PGG (0.1 and 5 mg/kg) lasted for 14 weeks. Viability was reduced by repeated PGG treatment in hMSC, preadipocytes, and cells under differentiation. PGG mainly induces apoptosis, and this effect is independent of its insulin mimetic action. In vivo, administration of ß-PGG attenuated shortening of the colon, hyperlipidaemia, fat cells and islet hypertrophy in DIO mice. Hepatic steatosis and related gene expression were improved along with glucose intolerance. Increased serum adiponectin, leptin, and glucagon-like peptide-1 levels were also observed. In conclusion, repeated PGG treatment interrupts the adipocyte lifecycle. PGG administration reduces adiposity and fatty liver development in DIO mice, and therefore, PGG could aid in clinical management of obesity.

Butyrate modulates adipose-derived stem cells isolated from polygenic obese and diabetic mice to drive enhanced immunosuppression.

BACKGROUND AIMS: Adipose-derived stem cells (ASCs) offer promising therapeutic possibilities for immunomodulation. Butyrate (BA) exerts potent anti-inflammatory effects and exhibits multiple regulatory functionalities in adipose tissue (AT). The authors aimed to explore whether BA modulates ASCs to augment their immunosuppressive capabilities. METHODS: The authors examined the potency of BA and ASCs for controlling anti-CD3 plus CD28-stimulated splenocyte proliferation in vitro, both in combination and with pre-treatment. Further, the authors investigated genes specifically upregulated by BA-treated ASCs, which were harvested from ASC-splenocyte co-culture after the removal of floating splenocytes. In addition, the authors investigated the influence of oral BA supplementation on the ex vivo immunosuppressive potency of ASCs from BALB/c and Tsumura, Suzuki, obese, diabetes (TSOD) mice. RESULTS: BA enhanced the immunosuppressive potency of ASCs when directly added to ASC-splenocyte co-cultures or via pre-conditioning treatment. The percentages of ASC-induced Foxp3+ regulatory T cells increased, whereas the numbers of ASC-suppressed T helper 17 cells further decreased after BA exposure. The messenger RNA expression levels of inducible nitric oxide (NO) synthase (iNOS), chemokines, IL-10 and amphiregulin in ASCs co-cultured with activated splenocytes were upregulated after incubation with BA. This was accompanied by an amplification of iNOS-inducing cytokines, interferon gamma and tumor necrosis factor alpha in the ASC-splenocyte co-culture, triggering ASCs to produce high NO levels under the influence of BA. Mechanistically, the authors detected BA-mediated acetylated histone H3 in ASCs. BA treatment consistently improved the immunosuppressive potency of ASCs derived from both BALB/c and TSOD mice. CONCLUSIONS: The use of BA to counteract metaflammation by restoring the defective immunomodulation of ASCs from dysregulated AT in obese donors is recommended.

Synthesis of Yakuchinone B-Inspired Inhibitors against Islet Amyloid Polypeptide Aggregation.

Type 2 diabetes mellitus (T2DM) is associated with pancreatic ß-cell dysfunction and insulin resistance. Islet amyloid polypeptide (IAPP) aggregation is found to induce islet ß-cell death in T2DM patients. Recently, we demonstrated that yakuchinone B derivative 1 exhibited inhibitory activity against IAPP aggregation. Thus, in this study, a series of synthesized yakuchinone B-inspired compounds were tested for their anti-IAPP aggregation activity. Four of these compounds, 4e-h, showed greater activity than the lead compound 1, in the sub-µM range (IC50 = 0.7-0.8 µM). The molecular docking analysis revealed crucial hydrogen bonds between the compounds and residues S19 and N22 and important hydrophobic interactions with residue I26. Notably, compounds 4g and 4h significantly protected ß-cells against IAPP-induced toxicity with EC50 values of 0.1 and 0.2 µM, respectively. In contrast, the protective activities of compounds 4e and 4f were weak. Overall, these results suggest that the compounds exhibiting IAPP aggregation-inhibiting activity have the potential to treat T2DM.

Investigation of Selected Flavonoid Derivatives as Potent FLT3 Inhibitors for the Potential Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC50 = 0.44 µM) but also FLT3-D835Y and FLT3-ITD mutants (IC50 = 0.23 and 0.39 µM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28, 31, 32, and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31, 32, and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.

Identification of a dual TAOK1 and MAP4K5 inhibitor using a structure-based virtual screening approach.

The STE20 kinase family is a complex signalling cascade that regulates cytoskeletal organisation and modulates the stress response. This signalling cascade includes various kinase mediators, such as TAOK1 and MAP4K5. The dysregulation of the STE20 kinase pathway is linked with cancer malignancy. A small-molecule inhibitor targeting the STE20 kinase pathway has therapeutic potential. In this study, a structure-based virtual screening (SBVS) approach was used to identify potential dual TAOK1 and MAP4K5 inhibitors. Enzymatic assays confirmed three potential dual inhibitors (>50% inhibition) from our virtual screening, and analysis of the TAOK1 and MAP4K5 binding sites indicated common interactions for dual inhibition. Compound 1 revealed potent inhibition of colorectal and lung cancer cell lines. Furthermore, compound 1 arrested cancer cells in the G0/G1 phase, which suggests the induction of apoptosis. Altogether, we show that the STE20 signalling mediators TAOK1 and MAP4K5 are promising targets for drug research.

Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei.

Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6‒16). Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 µM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 µM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.

Biological Evaluation of Selected Flavonoids as Inhibitors of MNKs Targeting Acute Myeloid Leukemia.

Excessive eIF4E phosphorylation by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNK1 and MNK2; collectively, MNKs) has been associated with oncogenesis. The overexpression of eIF4E in acute myeloid leukemia (AML) is related to cancer cell growth and survival. Thus, the inhibition of MNKs and eIF4E phosphorylation are potential therapeutic strategies for AML. Herein, a structure-based virtual screening approach was performed to identify potential MNK inhibitors from natural products. Three flavonoids, apigenin, hispidulin, and luteolin, showed MNK2 inhibitory activity with IC50 values of 308, 252, and 579 nM, respectively. A structure-activity relationship analysis was performed to disclose the molecular interactions. Furthermore, luteolin exhibited substantial inhibitory efficacy against MNK1 (IC50 = 179 nM). Experimental results from cellular assays showed that hispidulin and luteolin inhibited the growth of MOLM-13 and MV4-11 AML cells by downregulating eIF4E phosphorylation and arresting the cell cycle at the G0/G1 phase. Therefore, hispidulin and luteolin showed promising results as lead compounds for the potential treatment for AML.

Chalcones Display Anti-NLRP3 Inflammasome Activity in Macrophages through Inhibition of Both Priming and Activation Steps-Structure-Activity-Relationship and Mechanism Studies.

Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1ß, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1ß, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K+ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K+ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1ß. Next, 11Cha1 blocked ATP-mediated K+ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1ß and IL-18.

Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats.

N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids are components of lipofuscin and accumulate in retinal pigment epithelial (RPE) cells-these adducts are recognized in the pathogenesis of retinal degeneration. Further, blue light-emitting diode (LED) light (BLL)-induced retinal toxicity plays an important role in retinal degeneration. Here, we demonstrate that low-luminance BLL enhances phototoxicity in A2E-laden RPE cells and rats. RPE cells were subjected to synthetic A2E, and the effects of BLL on activation of apoptotic biomarkers were examined by measuring the levels of cleaved caspase-3. BLL modulates the protein expression of zonula-occludens 1 (ZO-1) and paracellular permeability in A2E-laden RPE cells. Early inflammatory and angiogenic genes were also screened after short-term BLL exposure. In this study, we developed a rat model for A2E treatment with or without BLL exposure for 21 days. BLL exposure caused fundus damage, decreased total retinal thickness, and caused neuron transduction injury in the retina, which were consistent with the in vitro data. We suggest that the synergistic effects of BLL and A2E accumulation in the retina increase the risk of retinal degeneration. These outcomes help elucidate the associations between BLL/A2E and angiogenic/apoptotic mechanisms, as well as furthering therapeutic strategies.

HADC8 Inhibitor WK2-16 Therapeutically Targets Lipopolysaccharide-Induced Mouse Model of Neuroinflammation and Microglial Activation.

Glial activation and neuroinflammatory processes play important roles in the pathogenesis of brain abscess and neurodegenerative diseases. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which contribute to the exacerbation of neuronal cell death. The inhibition of glial activation has been shown to alleviate neurodegenerative conditions. The present study was to investigate the specific HDAC8 inhibitor WK2-16, especially its effects on the neuroinflammatory responses through glial inactivation. WK2-16 significantly reduced the gelatinolytic activity of MMP-9, and expression of COX-2/iNOS proteins in striatal lipopolysaccharide (LPS)-induced neuroinflammation in C57BL/6 mice. The treatment of WK2-16 markedly improved neurobehavioral deficits. Immunofluorescent staining revealed that WK2-16 reduced LPS-stimulated astrogliosis and microglial activation in situ. Consistently, cellular studies revealed that WK2-16 significantly suppressed LPS-induced mouse microglia BV-2 cell proliferation. WK2-16 was proven to concentration-dependently induce the levels of acetylated SMC3 in microglial BV-2 cells. It also reduced the expression of COX-2/iNOS proteins and TNF-α production in LPS-activated microglial BV-2 cells. The signaling studies demonstrated that WK2-16 markedly inhibited LPS-activated STAT-1/-3 and Akt activation, but not NF-κB or MAPK signaling. In summary, the HADC8 inhibitor WK2-16 exhibited neuroprotective effects through its anti-neuroinflammation and glial inactivation properties, especially in microglia in vitro and in vivo.

A histone deacetylase inhibitor enhances expression of genes inhibiting Wnt pathway and augments activity of DNA demethylation reagent against nonsmall-cell lung cancer.

Development of new inhibitors targeting histone deacetylases (HDACs) with improved efficacy for solid tumor therapy is urgently needed. Here, we report the development of a novel HDAC inhibitor TMU-35435 and verify it as a single agent and in combination treatment with DNA demethylation reagent 5-aza-2'-deoxycytidine (5-aza-dC) in lung cancer preclinical models. TMU-35435 exerted cancer-specific cytotoxicity via mitochondria-mediated apoptosis. Expression microarrays revealed a unique TMU-35435-induced gene networks enriched in biological processes, including "negative regulation of cell proliferation" and "Wnt receptor signaling pathway" compared to FDA-approved HDAC inhibitor SAHA. TMU-35435 inhibited tumor growth with good pharmacokinetic properties and safety features in lung orthotopic and subcutaneously implanted xenograft models. TMU-35435 and 5-aza-dC showed synergistic antitumor effects through reactivation of tumor suppressor genes and those genes encoding negative regulators of Wnt signaling pathway in vitro and in vivo. Some genes showed additive inhibition of DNA methylation upon TMU-35435 and 5-aza-dC combined treatment. Our findings suggested that TMU-35435 is a potential HDAC inhibitor for lung cancer treatment as a single agent and in combination with 5-aza-dC.

The Novel HDAC8 Inhibitor WK2-16 Attenuates Lipopolysaccharide-Activated Matrix Metalloproteinase-9 Expression in Human Monocytic Cells and Improves Hypercytokinemia In Vivo.

Dysregulated human monocytes/macrophages can synthesize and secrete matrix metalloproteinases (MMPs), which play important roles in the progression of sepsis. In this study, we investigated the effects and mechanism of a novel histone deacetylase (HDAC8) inhibitor, (E)-N-hydroxy-4-methoxy-2-(biphenyl-4-yl)cinnamide (WK2-16), on MMP-9 production and activation in stimulated human monocytic THP-1 cells. Our results demonstrated that the acetylation level of structural maintenance of chromosomes 3 (SMC3) was up-regulated by WK2-16 in THP-1 cells. Consistently, an in vitro enzyme study demonstrated that WK2-16 selectively inhibited HDAC8 activity. Moreover, the WK2-16 concentration dependently suppressed MMP-9-mediated gelatinolysis induced by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). Additionally, WK2-16 significantly inhibited both MMP-9 protein and mRNA expression without cellular toxicity. Nevertheless, WK2-16 suppressed the extracellular levels of interleukin (IL)-6 from LPS-stimulated THP-1 cells. For the signaling studies, WK2-16 had no effect on LPS/TLR4 downstream signaling pathways, such as the NF-κB and ERK/JNK/P38 MAPK pathways. On the other hand, WK2-16 enhanced the recruitment of acetylated Yin Yang 1 (YY1) with HDAC1. Finally, in vivo studies indicated that WK2-16 could reduce the serum levels of TNF-α and IL-6 in endotoxemic mice. These results suggested that HDAC8 inhibition might provide a novel therapeutic strategy of hypercytokinemia in sepsis.

Total Synthesis and Metabolic Stability of Hispidulin and Its d-Labelled Derivative.

Hispidulin is a naturally occurring flavone known to have various Central nervous system (CNS) activities. Proposed synthetic approaches to synthesizing hispidulin have proven unsatisfactory due to their low feasibility and poor overall yields. To solve these problems, this study developed a novel scheme for synthesizing hispidulin, which had an improved overall yield as well as more concise reaction steps compared to previous methods reported. Additionally, using the same synthetic strategy, d-labelled hispidulin was synthesized to investigate its metabolic stability against human liver microsome. This work may produce new chemical entities for enriching the library of hispidulin-derived compounds.

Combination of the novel histone deacetylase inhibitor YCW1 and radiation induces autophagic cell death through the downregulation of BNIP3 in triple-negative breast cancer cells in vitro and in an orthotopic mouse model.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive and invasive of the breast cancer subtypes. TNBC is a challenging disease that lacks targets for treatment. Histone deacetylase inhibitors (HDACi) are a group of targeted anticancer agents that enhance radiosensitivity. Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a member of the Bcl-2 subfamily. BNIP3 is not found in normal breast tissue but is up-regulated in breast cancer. In the present study, we investigated the anti-cancer effects of a newly developed HDACi, YCW1, combined with ionizing radiation (IR) in TNBC in vitro and in an orthotopic mouse model. Furthermore, we examined the relationship between autophagy and BNIP3. METHODS: Trypan blue exclusion was used to investigate the viability of 4 T1 (a mouse TNBC cell line) and MDA-MB-231 cells (a human TNBC cell line) following combined YCW1 and IR treatment. Flow cytometry was used to determine apoptosis and autophagy. The expression levels of BNIP3, endoplasmic reticulum (ER) stress- and autophagic-related proteins were measured using western blot analysis. An orthotopic mouse model was used to investigate the in vivo effects of YCW1 and IR alone and in combination. Tumor volumes were monitored using a bioluminescence-based IVIS Imaging System 200. RESULTS: We found that YCW1 significantly enhanced toxicity in 4 T1 cells compared with suberoylanilide hydroxamic acid (SAHA), which was the first HDACi approved by the Food and Drug Administration for clinical use in cancer patients. The combined treatment of YCW1 and IR enhanced cytotoxicity by inducing ER stress and increasing autophagy induction. Additionally, the combined treatment caused autophagic flux and autophagic cell death. Furthermore, the expression level of BNIP3 was significantly decreased in cells following combined treatment. The downregulation of BNIP3 led to a significant increase in autophagy and cytotoxicity. The combined anti-tumor effects of YCW1 and IR were also observed in an orthotopic mouse model; combination therapy resulted in a significant increase in autophagy and decreased tumor tissue expression of BNIP3 in the tumor tissue. CONCLUSIONS: These data support the possibility of using a combination of HDACi and IR in the treatment of TNBC. Moreover, BNIP3 may be a potential target protein for TNBC treatment.

Synthesis and biological evaluation of lovastatin-derived aliphatic hydroxamates that induce reactive oxygen species.

Some hydroxamate compounds induce cancer cell death by intracellular reactive oxygen species (ROS). This study introduced the hydroxamate core into lovastatin, a fungus metabolite clinically used for the treatment of hypercholesterolemia. The resulting compounds were evaluated for the activity for inducing ROS production. Most compounds exhibited higher activity than original lovastatin. Of these compounds, compound 3c had the most potent activity. Test of cytotoxicity in a panel of human cancer cell lines indicated compound 3c had activities superior to cisplatin in prostate cancer PC-3 cells and breast cancer T47D cells. In contrast, it in amounts up to 40µM had a much lower cytotoxic effect on normal human IMR-90 cells. Further profiling of cell cycle progression, cell apoptosis, and DNA damage activated checkpoint signaling pathway revealed the important role of compound 3c-mediated cytotoxicity in ROS generation.

Total Synthesis of Hispidulin and the Structural Basis for Its Inhibition of Proto-oncogene Kinase Pim-1.

A new method is applied to synthesize hispidulin, a natural flavone with a broad spectrum of biological activities. Hispidulin exhibits inhibitory activity against the oncogenic protein kinase Pim-1. Crystallographic analysis of Pim-1 bound to hispidulin reveals a binding mode distinct from that of quercetin, suggesting that the binding potency of flavonoids is determined by their hydrogen-bonding interactions with the hinge region of the kinase. Overall, this work may facilitate construction of a library of hispidulin-derived compounds for investigating the structure-activity relationship of flavone-based Pim-1 inhibitors.