Design, synthesis, and anti-cancer evaluation of C-14 arylcarbamate derivatives of andrographolide.

In this study, we explored a concise and mild synthetic route to produce novel C-14 arylcarbamate derivatives of andrographolide, a known anti-inflammatory and anticancer natural product. Upon assessing their anti-cancer efficacy against pancreatic ductal adenocarcinoma (PDAC) cells, some derivatives showed stronger cytotoxicity against PANC-1 cells than andrographolide. In addition, we demonstrated one derivative, compound 3m, effectively reduced the expression of oncogenic p53 mutant proteins (p53R273H and p53R248W), proliferation, and migration in PDAC lines, PANC-1 and MIA PaCa-2. Accordingly, the novel derivative holds promise as an anti-cancer agent against pancreatic cancer. In summary, our study broadens the derivative library of andrographolide and develops an arylcarbamate derivative of andrographolide with promising anticancer activity against PDAC.

Galgravin Isolated from Piper kadsura Ameliorates Lipopolysaccharide (LPS)-Induced Endotoxemia in Mice.

Sepsis results from uncontrolled inflammation, characterized by cytokine storm and immunoparalysis. To assess whether galgravin, a natural lignan isolated from Piper kadsura, can be used to treat sepsis, models of bacterial lipopolysaccharide (LPS)-activated macrophages and LPS-induced endotoxemia mice were used. Galgravin suppressed NF-κB activation in LPS-activated RAW 264.7 macrophages without causing significant cytotoxicity, in which proinflammatory molecules like TNF-α, IL-6, iNOS, and COX-2 were downregulated. In addition, the expression of TNF-α and IL-6 was also suppressed by galgravin in LPS-activated murine bone marrow-derived macrophages. Moreover, galgravin significantly downregulated the mRNA expression of TNF-α, IL-6, and iNOS in the lungs and decreased TNF-α and IL-6 in the serum and IL-6 in the bronchoalveolar lavage fluid of LPS-challenged mice. The COX-2 expression in tissues, including the lung, liver, and kidney, as well as the lung alveolar hemorrhage, was also reduced by galgravin. The present study reveals the anti-inflammatory effects of galgravin in mouse models and implies its potential application in inflammation diseases.

Andrographolide suppresses the malignancy of triple-negative breast cancer by reducing THOC1-promoted cancer stem cell characteristics.

Triple-negative breast cancers (TNBCs) are difficult to cure and currently lack of effective treatment strategies. Cancer stem cells (CSCs) are highly associated with the poor clinical outcome of TNBCs. Thoc1 is a core component of the THO complex (THOC) that regulates the elongation, processing and nuclear export of mRNA. The function of thoc1 in TNBC and whether Thoc1 serves as a drug target are poorly understood. In this study, we demonstrated that thoc1 expression is elevated in TNBC cell lines and human TNBC patient tissues. Knockdown of thoc1 decreased cancer stem cell populations, reduced mammosphere formation, impaired THOC function, and downregulated the expression of stemness-related proteins. Moreover, the thoc1-knockdown 4T1 cells showed less lung metastasis in an orthotopic breast cancer mouse model. Overexpression of Thoc1 promoted TNBC malignancy and the mRNA export of stemness-related genes. Furthermore, treatment of TNBC cells with the natural compound andrographolide reduced the expression of Thoc1 expression, impaired homeostasis of THOC, suppressed CSC properties, and delayed tumor growth in a 4T1-implanted orthotopic mouse model. Andrographolide also reduced the activity of NF-κB, an upstream transcriptional regulator of Thoc1. Notably, thoc1 overexpression attenuates andrographolide-suppressed cellular proliferation. Altogether, our results demonstrate that THOC1 promotes cancer stem cell characteristics of TNBC, and andrographolide is a potential natural compound for eliminating CSCs of TNBCs by downregulating the NF-κB-thoc1 axis.

A Houttuynia cordata-based Chinese herbal formula improved symptoms of allergic rhinitis during the COVID-19 pandemic.

BACKGROUND: The symptoms of coronavirus disease 2019 (COVID-19) such as hyposmia, rhinorrhea, nasal obstruction, and cough are similar to those of chronic allergic rhinitis (AR). Such symptoms can easily lead AR patients to unnecessary anxiety, misdiagnosis, and invasive diagnostic tests in the COVID-19 pandemic. Interleukin-6 (IL-6) is an important mediator for chronic AR and plays a crucial role in the inflammation of COVID-19. Houttuynia cordata (HC) has been shown to reduce nasal congestion and swelling by suppressing the activation of IL-6 and is used to fight COVID-19. A novel HC-based Chinese herbal formula, Zheng-Yi-Fang (ZYF), was developed to test effects on nasal symptoms of patients with AR in the COVID-19 pandemic. METHODS: Participants aged between 20 and 60 years with at least a 2-year history of moderate to severe perennial AR were enrolled. Eligible participants were randomly allocated to either the intervention group (taking ZYF) or the control group (using regular western medicine) for 4 weeks. The Chinese version of the Rhinosinusitis Outcome Measures was used to evaluate impacts on quality of life and nasal symptoms of participants with AR. In addition, the effect of ZYF on lipopolysaccharide (LPS)-induced IL-6 was investigated. RESULTS: Participants with AR taking ZYF improved their symptoms of nasal obstruction, nasal secretion, hyposmia, and postnasal drip in comparison with those of the control group. Meanwhile, ZYF exhibited inhibition of IL-6 secretion in the LPS-induced inflammatory model. CONCLUSION: ZYF has potential effects to relieve nasal symptoms for AR during the COVID-19 pandemic.

Perturbation of p38α MAPK as a Novel Strategy to Effectively Sensitize Chronic Myeloid Leukemia Cells to Therapeutic BCR-ABL Inhibitors.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the presence of the BCR-ABL oncogene. Therapeutic regimens with tyrosine kinase inhibitors (TKIs) specifically targeting BCR-ABL have greatly improved overall survival of CML. However, drug intolerance and related toxicity remain. Combined therapy is effective in reducing drug magnitude while increasing therapeutic efficacy and, thus, lowers undesired adverse side effects. The p38 MAPK activity is critically linked to the pathogenesis of a number of diseases including hematopoietic diseases; however, the role of each isozyme in CML and TKI-mediated effects is still elusive. In this study, we used specific gene knockdown to clearly demonstrate that the deficiency of p38α greatly enhanced the therapeutic efficacy in growth suppression and cytotoxicity of TKIs, first-generation imatinib, and second generation dasatinib by approximately 2.5-3.0-fold in BCR-ABL-positive CML-derived leukemia K562 and KMB5 cells. Knockdown of p38ß, which displays the most sequence similarity to p38α, exerted distinct and opposite effects on the TKI-mediated therapeutic efficacy. These results show the importance of isotype-specific intervention in enhancing the therapeutic efficacy of TKI. A highly specific p38α inhibitor, TAK715, also significantly enhanced the imatinib- and dasatinib-mediated therapeutic efficacy, supporting the feasibility of p38α deficiency in future clinic application. Taken together, our results demonstrated that p38α is a promising target for combined therapy with BCR-ABL-targeting tyrosine kinase inhibitors for future application to increase therapeutic efficacy.

Arginine Methylation of hnRNPK Inhibits the DDX3-hnRNPK Interaction to Play an Anti-Apoptosis Role in Osteosarcoma Cells.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA/DNA binding protein involved in diverse cell processes; it is also a p53 coregulator that initiates apoptosis under DNA damage conditions. However, the upregulation of hnRNPK is correlated with cancer transformation, progression, and migration, whereas the regulatory role of hnRNPK in cancer malignancy remains unclear. We previously showed that arginine methylation of hnRNPK attenuated the apoptosis of U2OS osteosarcoma cells under DNA damage conditions, whereas the replacement of endogenous hnRNPK with a methylation-defective mutant inversely enhanced apoptosis. The present study further revealed that an RNA helicase, DDX3, whose C-terminus preferentially binds to the unmethylated hnRNPK and could promote such apoptotic enhancement. Moreover, C-terminus-truncated DDX3 induced significantly less apoptosis than full-length DDX3. Notably, we also identified a small molecule that docks at the ATP-binding site of DDX3, promotes the DDX3-hnRNPK interaction, and induces further apoptosis. Overall, we have shown that the arginine methylation of hnRNPK suppresses the apoptosis of U2OS cells via interfering with DDX3-hnRNPK interaction. On the other hand, DDX3-hnRNPK interaction with a proapoptotic role may serve as a target for promoting apoptosis in osteosarcoma cells.

Anti-Cancer and Anti-Inflammatory Activities of Three New Chromone Derivatives from the Marine-Derived Penicillium citrinum.

Three new and uncommon chromone analogs, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), were isolated from marine-origin Penicillium citrinum. Among the isolated compounds, compounds 2-3 remarkably suppressed fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 31.68 ± 2.53, and 33.52 ± 0.42 µM, respectively. Compound 3 exhibited cytotoxic activities against human colon carcinoma (HT-29) and non-small lung cancer cell (A549) with IC50 values of 21.17 ± 4.89 and 31.43 ± 3.01 µM, respectively, and Western blot assay confirmed that compound 3 obviously induced apoptosis of HT-29 cells, via Bcl-2, Bax, and caspase 3 signaling cascades.

Rare Chromone Derivatives from the Marine-Derived Penicillium citrinum with Anti-Cancer and Anti-Inflammatory Activities.

Three new and rare chromone derivatives, epiremisporine C (1), epiremisporine D (2), and epiremisporine E (3), were isolated from marine-derived Penicillium citrinum, together with four known compounds, epiremisporine B (4), penicitrinone A (5), 8-hydroxy-1-methoxycarbonyl-6-methylxanthone (6), and isoconiochaetone C (7). Among the isolated compounds, compounds 2-5 significantly decreased fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 6.39 ± 0.40, 8.28 ± 0.29, 3.62 ± 0.61, and 2.67 ± 0.10 µM, respectively. Compounds 3 and 4 exhibited cytotoxic activities with IC50 values of 43.82 ± 6.33 and 32.29 ± 4.83 µM, respectively, against non-small lung cancer cell (A549), and Western blot assay confirmed that compounds 3 and 4 markedly induced apoptosis of A549 cells, through Bcl-2, Bax, and caspase 3 signaling cascades.

Evaluation of Antioxidant and Anti-α-glucosidase Activities of Various Solvent Extracts and Major Bioactive Components from the Seeds of Myristica fragrans.

Myristica fragrans is a well-known species for flavoring many food products and for formulation of perfume and medicated balm. It is also used to treat indigestion, stomach ulcers, liver disorders, and, as emmenagogue, diaphoretic, diuretic, nervine, and aphrodisiac. We examined antioxidant properties and bioactive compounds in various solvent extracts from the seeds of M. fragrans. Methanol, ethanol, and acetone extracts exhibited relatively strong antioxidant activities by 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), superoxide radical, and hydroxyl radical scavenging tests. Furthermore, methanol extracts also displayed significant anti-α-glucosidase activity. Examined and compared to the various solvent extracts for their chemical compositions using HPLC analysis, we isolated the ten higher content compounds and analyzed antioxidant and anti-α-glucosidase activities. Among the isolates, dehydrodiisoeugenol, malabaricone B and malabaricone C were main antioxidant components in seeds of M. fragrans. Malabaricone C exhibited stronger antioxidant capacities than others based on lower half inhibitory concentration (IC50) values in DPPH and ABTS radical scavenging assays, and it also showed significant inhibition of α-glucosidase. These results shown that methanol was found to be the most efficient solvent for extracting the active components from the seeds of M. fragrans, and this material is a potential good source of natural antioxidant and α-glucosidase inhibitor.

Andrographolide and its fluorescent derivative inhibit the main proteases of 2019-nCoV and SARS-CoV through covalent linkage.

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested.

Vaccine adjuvant activity of a TLR4-activating synthetic glycolipid by promoting autophagy.

Toll-like receptors (TLRs) play crucial roles in host immune defenses. Recently, TLR-mediated autophagy is reported to promote immune responses via increasing antigen processing and presentation in antigen presenting cells. The present study examined whether the synthetic TLR4 activator (CCL-34) could induce autophagy to promote innate and adaptive immunity. In addition, the potential of CCL-34 as an immune adjuvant in vivo was also investigated. Our data using RAW264.7 cells and bone marrow-derived macrophages showed that CCL-34 induced autophagy through a TLR4-NF-κB pathway. The autophagy-related molecules (Nrf2, p62 and Beclin 1) were activated in RAW264.7 cells and bone marrow-derived macrophages under CCL-34 treatment. CCL-34-stimulated macrophages exhibited significant antigen-processing activity and induced the proliferation of antigen-specific CD4+T cells as well as the production of activated T cell-related cytokines, IL-2 and IFN-γ. Furthermore, CCL-34 immunization in mice induced infiltration of monocytes in the peritoneal cavity and elevation of antigen-specific IgG in the serum. CCL-34 treatment in vivo did not cause toxicity based on serum biochemical profiles. Notably, the antigen-specific responses induced by CCL-34 were attenuated by the autophagy inhibitor, 3-methyladenine. In summary, we demonstrated CCL-34 can induce autophagy to promote antigen-specific immune responses and act as an efficient adjuvant.

hnRNPK S379 phosphorylation participates in migration regulation of triple negative MDA-MB-231 cells.

We have previously identified a novel Aurora-A-mediated Serine 379 (S379) phosphorylation of a poly(C)-binding protein, hnRNPK, the overexpression of which is frequently observed in various cancers. It is known that the oncogenic Aurora-A kinase promotes the malignancy of cancer cells. This study aims to investigate the unexplored functions of hnRNPK S379 phosphorylation using MDA-MB-231 cells, a triple negative breast cancer cell that has amplification of the Aurora-A kinase gene. Accordingly, we established two cell lines in which the endogenous hnRNPK was replaced with either S379D or S379A hnRNPK respectively. Notably, we found that a phosphorylation-mimic S379D mutant of hnRNPK suppressed cell migration and, conversely, a phosphorylation-defective S379A mutant promoted migration. Moreover, Twist was downregulated upon hnRNPK S379 phosphorylation, whereas ß-catenin and MMP12 were increased when there was loss of hnRNPK S379 phosphorylation in MDA-MB-231 cells. Furthermore, S379A hnRNPK increases stability of ß-catenin in MDA-MB-231 cells. In conclusion, our results suggest that hnRNPK S379 phosphorylation regulates migration via the EMT signaling pathway.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein.

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

Pilloin, A Flavonoid Isolated from Aquilaria sinensis, Exhibits Anti-Inflammatory Activity In Vitro and In Vivo.

Flavonoids, widely present in medicinal plants and fruits, are known to exhibit multiple pharmacological activities. In this study, we isolated a flavonoid compound, pilloin, from Aquilaria sinensis and investigated its anti-inflammatory activity in bacterial lipopolysaccharide-induced RAW 264.7 macrophages and septic mice. Pilloin inhibited NF-κB activation and reduced the phosphorylation of IκB in LPS-stimulated macrophages. Moreover, pilloin significantly suppressed the production of pro-inflammatory molecules, such as TNF-α, IL-6, COX-2 and iNOS, in LPS-treated RAW 264.7 macrophages. Additionally, pilloin suppressed LPS-induced morphological alterations, phagocytic activity and ROS elevation in RAW 264.7 macrophages. The mitogen-activated protein kinase-mediated signalling pathways (including JNK, ERK, p38) were also inhibited by pilloin. Furthermore, pilloin reduced serum levels of TNF-α (from 123.3 ± 7 to 46.6 ± 5.4 ng/mL) and IL-6 levels (from 1.4 ± 0.1 to 0.7 ± 0.1 ng/mL) in multiple organs of LPS-induced septic mice (liver: from 71.8 ± 3.2 to 36.7 ± 4.3; lung: from 118.6 ± 10.6 to 75.8 ± 11.9; spleen: from 185.9 ± 23.4 to 109.6 ± 18.4; kidney: from 160.3 ± 11.8 to 75 ± 10.8 pg/mL). In summary, our results demonstrate the anti-inflammatory potential of pilloin and reveal its underlying molecular mechanism of action.

Andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.

BACKGROUND: Activation of Ras oncogene in human tumors is associated with radiation-associated metastatic potential. Although ionizing radiation is one important method of cancer treatments, it has been shown to enhance matrix metalloproteinases (MMPs) activity and facilitates a more aggressive cancer phenotype. Our previous studies showed that andrographolide with lower dose rates of radiation could inhibit RAS-transformed cancer metastasis in vivo; however, the molecular mechanisms are not yet clear. In this study, we aimed to explore the anti-metastatic effect of andrographolide combined with radiation on Ras-transformed cells. METHODS: RAS-transformed cells were treated with andrographolide in the presence or absence of irradiation (2-4 Gy) or angiotensin II to examine cell invasion. In vivo tumorigenesis assays were also performed. The MMP-2 activity was detected by using Gelatin zymography. Signal transduction of NF-κB subunit, p65 and phosphor-ERK 1/2, were examined by using Western blotting analysis. RESULTS: Treatment with andrographolide inhibited migration of Ras-transformed cells. Andrographolide treatment with radiation significantly inhibited cancer metastasis in vivo. We found that andrographolide exhibited anti-migration and anti-invasive ability against cancer metastasis via inhibition of MMP2 activity rather than affected MMP-9 and EMT. In addition, combined andrographolide with radiation appeared to be more effective in reducing MMP-2 expression, and this effect was accompanied by suppression of ERK activation that inhibits cancer cell migration and invasion. CONCLUSIONS: These findings suggest that andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.

Chinese herbal products and the reduction of risk of breast cancer among females with type 2 diabetes in Taiwan: A case-control study.

Women with type 2 diabetes have a higher risk of developing breast cancer. In Taiwan, traditional Chinese medicine is widely used to treat diabetes; however, its precise influence has not been empirically tested.The objective of the case-control study is to estimate the effect on the risk of breast cancer of using traditional Chinese medicine for women with type 2 diabetes.More than 80% of women with type 2 diabetes have used traditional Chinese medicine. The most commonly prescribed drug was metformin and the herbal formulas were the Di Huang Wan series, including Liu Wei Di Huang Wan, Qi Ju Di Huang Wan, and Zhi Bai Di Huang Wan, followed by Bai Hu Jia Ren Shen Tang and Yu Quan Wan. For the effect of metformin, women who used traditional Chinese medicine including Di Huang series have a lower risk of breast cancer HR: 0.35 (95%CI: 0.23-0.51) in women younger than 55 and HR: 0.54 (95%CI: 0.37-0.79) in women older than 55.The protective effect of the Di Huang Wan series may be due to the synergetic effect of reducing blood glucose or increasing insulin sensitivity and delaying the insulin resistance of cells. The relationship between the Di Huang Wan series and breast cancer of women with type 2 diabetes requires further investigation.

2-(2-Phenylethyl)-4H-chromen-4-one Derivatives from the Resinous Wood of Aquilaria sinensis with Anti-Inflammatory Effects in LPS-Induced Macrophages.

The resinous wood of Aquilaria sinensis, known as agarwood (Chen Xiang in Chinese), is traditionally used for the treatment of abdominal pain, vomiting, circulatory disorders, and dyspnea. Four new 2-(2-phenylethyl)-4H-chromen-4-one derivatives, namely 7-methoxy-2-[2-(4'-hydroxy-phenyl)ethyl]chromone (1), 7-hydroxy-2-[2-(4'-methoxyphenyl)ethyl]chromone (2), 5,6-dihydroxy- 2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]chromone (3), and 6-hydroxy-5-methoxy-2-(2-phenyl-ethyl)chromone (4), have been isolated from the resinous wood of A. sinensis, together with nine known compounds. The structures of these compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, neopetasan, 7-methoxy-2-(2-phenylethyl)-chromone, 6,7-dimethoxy-2-(2-phenylethyl)chromone, and 6,7-dimethoxy-2-[2-(4'-methoxy-phenyl)ethyl]chromone inhibited NF-κB activation in LPS-stimulated RAW 264.7 macrophages with relative luciferase activity values of 0.55 ± 0.09, 0.54 ± 0.03, 0.31 ± 0.05, and 0.38 ± 0.14, respectively, versus that of vehicle control (1.03 ± 0.02). In addition, 5,6-dihydroxy-2-[2-(3'-hydroxy-4'-methoxyphenyl)ethyl]chromone, 7-methoxy-2-(2-phenylethyl)chromone, 7-dimethoxy-2-(2-phenylethyl)chromone, and 6,7-dimethoxy-2-[2-(4'-methoxyphenyl)ethyl]chromone could suppress LPS-induced NO production in RAW 264.7 cells and did not induce cytotoxicity against RAW 264.7 cells after 24-h treatment.

Anti-inflammatory Principles from Sarcandra glabra.

Sarcandra glabra (Thunb.) Nakai (Chloranthaceae) is a medicinal plant used as herbal tea or food supplement to promote human health. We isolated 14 phenolic compounds from the n-butanol fraction of S. glabra and investigated their anti-inflammatory potential using lipopolysaccharide (LPS)-activated RAW264.7 macrophages. We demonstrated that methyl isorinate, a previously uncharacterized compound in S. glabra, is able to suppress NF-κB activation and reduce the expression of iNOS and COX-2 as well as the phosphorylation of IκB in LPS-treated RAW264.7 cells. In addition, the production of two inflammatory cytokines (IL-6 and TNF-α), as well as release of reactive oxygen species, in the LPS-stimulated macrophages was also inhibited by this compound. Furthermore, the structure-activity relationships of all of the isolated phenolic compounds present were analyzed. Overall, this study revealed several anti-inflammatory compounds that were present in S. glabra, and the results suggest that these diverse phenolic compounds are associated with the anti-inflammatory effects of S. glabra.

Development of a Bifunctional Andrographolide-Based Chemical Probe for Pharmacological Study.

Andrographolide (ANDRO) is a lactone diterpenoid compound present in the medicinal plant Andrographis paniculata which is clinically applied for multiple human diseases in Asia and Europe. The pharmacological activities of andrographolide have been widely demonstrated, including anti-inflammation, anti-cancer and hepatoprotection. However, the pharmacological mechanism of andrographolide remains unclear. Therefore, further characterization on the kinetics and molecular targets of andrographolide is essential. In this study, we described the synthesis and characterization of a novel fluorescent andrographolide derivative (ANDRO-NBD). ANDRO-NBD exhibited a comparable anti-cancer spectrum to andrographolide: ANDRO-NBD was cytotoxic to various types of cancer cells and suppressed the migration activity of melanoma cells; ANDRO-NBD treatment induced the cleavage of heat shock protein 90 (Hsp90) and the downregulation of its client oncoproteins, v-Src and Bcr-abl. Notably, ANDRO-NBD showed superior inhibitory effects to andrographolide in all anticancer assays we have performed. In addition, ANDRO-NBD was further used as a fluorescent probe to investigate the uptake kinetics, cellular distribution and molecular targets of andrographolide. Our data revealed that ANDRO-NBD entered cells rapidly and its fluorescent signal could be detected in nucleus, cytoplasm, mitochondria, and lysosome. Moreover, we demonstrated that ANDRO-NBD was covalently bound to several putative target proteins of andrographolide, including NF-κB and hnRNPK. In summary, we developed a fluorescent andrographolide probe with comparable bioactivity to andrographolide, which serves as a powerful tool to explore the pharmacological mechanism of andrographolide.

CCL-34, a synthetic toll-like receptor 4 activator, modulates differentiation and maturation of myeloid dendritic cells.

CCL-34, a synthetic α-galactosylceramide analog, has been reported as an activator of toll-like receptor 4 (TLR4) in macrophages. TLR4 is highly expressed in dendritic cell (DC) and several TLR4 agonists are known to trigger DC maturation. We herein evaluated the effect of CCL-34 on DC maturation. Human CD14+ monocyte-derived immature DC were treated with CCL-34, its inactive structural analog CCL-44, or LPS to assess the DC maturation. CCL-34 induced DC maturation according to their characteristically dendrite-forming morphology, CD83 expression and IL-12p70 production. The allostimulatory activity of DC on proliferation of naive CD4+CD45+RA+ T cells and their secretion of interferon-γ was increased by CCL-34. Phagocytosis, an important function of immature DC, was reduced after CCL-34 treatment. All these effects related to DC maturation were evidently induced by positive control LPS but not by CCL-44 treatment. TLR4 neutralization impaired human DC maturation triggered by CCL-34. The induction of IL-12, a hallmark of DC maturation, by CCL-34 and LPS was only evident in TLR4-competent C3H/HeN, but not in TLR4-defective C3H/HeJ mice. CCL-34 could further elicit the antigen presentation capability in mice inoculated with doxorubicin-treated colorectal cancer cells. In summary, CCL-34 triggers DC maturation via a TLR4-dependent manner, which supports its potential application as an immunostimulator.