Anti-Inflammatory and Anti-Adipocyte Dysfunction Effects of Ficus lindsayana Latex and Root Extracts.

Low-grade chronic inflammation and adipocyte dysfunction are prominent risk factors of insulin resistance and type 2 diabetes mellitus (T2DM) in obesity. Thus, prevention of inflammation and adipocyte dysfunction could be one possible approach to mitigate T2DM development. Several Ficus species have been used in traditional medicine for ameliorating inflammation and T2DM. Our previous studies reported biological effects of Ficus lindsayana including antioxidant, anti-cancer, and anti-α-glucosidase activities. Further, this study therefore investigated whether F. lindsayana latex (FLLE) and root (FLRE) extracts inhibit inflammation-stimulated insulin resistance in adipocytes and inflammation in macrophages. FLLE and FLRE (200 µg/mL) had no significant cytotoxicity for macrophages, adipocytes, and blood cells (PBMCs and RBCs). FLRE had a total flavonoid content about three times higher than FLLE, while both had similar levels of total phenolic content. FLRE showed higher abilities than FLLE in suppressing inflammation in both macrophages and adipocytes and reversing the inflammation-induced insulin resistance in adipocytes. In TNF-α-induced adipocytes, FLRE significantly improved insulin-induced glucose uptake and insulin-suppressed lipolysis, while FLLE only significantly improved glucose uptake. Moreover, FLRE and FLLE remarkably reduced chemoattractant (MCP-1) but improved adipogenic (PPARγ and CEBPα) gene expression, leading to the promotion of adipogenesis and the suppression of insulin resistance. In LPS-induced macrophages, FLRE, but not FLLE, significantly inhibited LPS-induced NO production. Moreover, FLRE significantly reduced LPS-stimulated iNOS, COX-2, IL-1ß, IL-6, and TNF-α gene expression. These results may provide the potential data for the development of this plant, especially the root part, as an alternative medicine, functional ingredient, or food supplement for the prevention of inflammation and obesity-associated insulin resistance, as well as T2DM.

Antidiabetic effects of Andrographis paniculata supplementation on biochemical parameters, inflammatory responses, and oxidative stress in canine diabetes.

Introduction: Diabetes mellitus is a common endocrine disorder that causes hyperglycemia in dogs. Persistent hyperglycemia can induce inflammation and oxidative stress. This study aimed to investigate the effects of A. paniculata (Burm.f.) Nees (Acanthaceae) (A. paniculata) on blood glucose, inflammation, and oxidative stress in canine diabetes. A total of 41 client-owned dogs (23 diabetic and 18 clinically healthy) were included in this double-blind, placebo-controlled trial. Methods: The diabetic dogs were further divided into two treatments protocols: group 1 received A. paniculata extract capsules (50 mg/kg/day; n = 6) or received placebo for 90 days (n = 7); and group 2 received A. paniculata extract capsules (100 mg/kg/day; n = 6) or received a placebo for 180 days (n = 4). Blood and urine samples were collected every month. No significant differences in fasting blood glucose, fructosamine, interleukin-6, tumor necrosis factor-alpha, superoxide dismutase, and malondialdehyde levels were observed between the treatment and placebo groups (p > 0.05). Results and Discussion: The levels of alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, and creatinine were stable in the treatment groups. The blood glucose levels and concentrations of inflammatory and oxidative stress markers in the client-owned diabetic dogs were not altered by A. paniculata supplementation. Furthermore, treatment with this extract did not have any adverse effects on the animals. Non-etheless, the effects of A. paniculata on canine diabetes must be appropriately evaluated using a proteomic approach and involving a wider variety of protein markers.

Stabilization of Antioxidant and Anti-Inflammatory Activities of Nano-Selenium Using Anoectochilus burmannicus Extract as a Potential Novel Functional Ingredient.

Anoectochilus burmannicus is an orchid that contains phenolic compounds and exhibits antioxidant and anti-inflammation properties. This study aimed to investigate whether its ethanolic extract (ABE) can be used as a reducing agent and/or a stabilizer of nano-selenium (SeNP) synthesis. SeNPs exhibited higher antioxidant activity than ABE-SeNPs. In contrast, ABE-SeNP (4 µM Se) had greater anti-inflammatory activity in LPS-induced macrophages than SeNPs. Interestingly, ABE acted as a stabilizer for SeNPs by preventing particle aggregation and preserving its antioxidant activity after long-term storage (90 days). Moreover, after the freeze-drying process, ABE-SeNPs could be completely reconstituted to suspension with significantly stable antioxidant and anti-inflammatory activities compared to freshly prepared particles, suggesting the cryoprotectant and/or lyoprotectant role of ABE. The present study shows the potential of ABE as an effective stabilizer for nanoparticles and provides evidence for the development of ABE-SeNPs as a food supplement or novel functional ingredient for health benefits.

Ficus dubia latex extract prevent DMH-induced rat early colorectal carcinogenesis through the regulation of xenobiotic metabolism, inflammation, cell proliferation and apoptosis.

Ficus dubia latex is recognized as a remedy in Asian traditional medicine with various therapeutic effects. The present study aimed to determine the preventive action of Ficus dubia latex extract (FDLE) on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis and its mechanisms. The experiment included an initiation model in which rats were orally administered with FDLE daily for 1 week before DMH injection until the end of the experiment, while only after DMH injection until the end in the post-initiation model. The results firstly indicated that FDLE treatment could reduce the level of methylazoxymethanol (MAM) in rat colonic lumen by inhibition of the activities of both phase I xenobiotic metabolizing enzymes in the liver and ß-glucuronidase in the colon, leading to reduced DNA methylation in colonic mucosal cells, related to the number of ACF in the initiation stage. Besides, FDLE modulated the inflammation which could suppress the growth and induce apoptosis of aberrant colonic mucosal cells, leading to retardation of ACF multiplicity. Therefore, FDLE showed the ability to suppress the DMH-induced rat ACF formation and inflammation promoted growth of ACF. In conclusion, FDLE had the potential to prevent carcinogens-induced rat colorectal carcinogenesis in the initiation stage.

Ficus dubia Latex Extract Induces Cell Cycle Arrest and Apoptosis by Regulating the NF-κB Pathway in Inflammatory Human Colorectal Cancer Cell Lines.

Colorectal cancer is one of the most diagnosed cancers that is associated with inflammation. Ficus dubia latex is recognized as a remedy with various therapeutic effects in traditional medicine, including anti-inflammatory and antioxidant activity. The present study aims to compare the anti-tumor activity of Ficus dubia latex extract (FDLE) against HCT-116 and HT-29 human colorectal cancer cell lines in normal and inflammatory condition and explore its mechanism of action. FDLE exhibited remarkable antiproliferative activity against HCT-116 and HT-29 colorectal cancer cell lines in both conditions using MTT and colony formation assays and more effective anti-proliferation was observed in inflammatory condition. Mechanistically, FDLE induced cell cycle arrest at G0/G1 phase by down-regulating NF-κB, cyclin D1, CDK4 and up-regulatingp21 in both cell in normal condition. In inflammatory condition, FDLE not only exhibited stronger induction of cell cycle arrest in both cells by down-regulating NF-κB, cyclin D1, CDK4 and down-regulating p21, but also selectively induced apoptosis in HCT-116 cells by down-regulating NF-κB and Bcl-xl and up-regulating Bid, Bak, cleaved caspase-7 and caspase-3 through stronger ability to regulate these proteins. Our results demonstrated that the phytochemical agent in the latex of Ficus dubia could potential be used for treatment and prevention of human colorectal cancer, especially in inflammation-induced hyperproliferation progression.

Phikud Navakot extract attenuates lipopolysaccharide-induced inflammatory responses through inhibition of ERK1/2 phosphorylation in a coculture system of microglia and neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Phikud Navakot (PN), a mixture of nine herbal plants, is an ancient Thai traditional medicine used for relieving circulatory disorders and dizziness. PN has also shown anti-inflammatory effects in rats with acute myocardial infarction. Moreover, phytochemical-inhibiting neuroinflammation, including gallic acid, vanillic acid, ferulic acid, and rutin were detected in PN extract; however, the anti-neuroinflammatory activity of PN extract and its components in a coculture system of microglia and neuronal cells is limited. OBJECTIVE: To investigate the anti-neuroinflammatory activities of PN on lipopolysaccharide (LPS)-induced inflammation in a coculture system of microglia and neuronal cells. METHODS: ELISA and qRT-PCR were used to assess cytokine expression. The phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting. Microglia-mediated neuroinflammation was evaluated using a BV-2 microglia-N2a neuron transwell co-culture. RESULTS: PN extract and its component, gallic acid, decreased LPS-induced the mRNA expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as well as IL-6 protein levels in both microglial monoculture and coculture systems. This was accompanied by a reduction in neurodegeneration triggered by microglia in N2a neurons with increased neuronal integrity markers (ßIII tubulin and tyrosine hydroxylase (TH)). These effects were caused by the ability of PN extract to inhibit extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. CONCLUSION: This is the first study to show that PN extract inhibits neurodegeneration in LPS-activated BV-2 microglia by targeting ERK signaling activity.

Ethanolic Fruit Extract of Emblica officinalis Suppresses Neuroinflammation in Microglia and Promotes Neurite Outgrowth in Neuro2a Cells.

Inhibiting neuroinflammation and modulating neurite outgrowth could be a promising strategy to prevent neurological disorders. Emblica officinalis (EO) may be a potent agent against them. Although EO extract reportedly has anti-inflammatory properties in macrophages, there is limited knowledge about its neuroprotective activity by suppressing microglia-mediated proinflammatory cytokine production and inducing neurite outgrowth. The present study aimed to elucidate the effect of EO fruit extract on the lipopolysaccharide- (LPS-) induced neuroinflammation using microglial (BV2) and neuroblastoma (Neuro2a) cells. The results demonstrated that, in LPS-treated BV2 cells, EO fruit extract reduced nitric oxide, interleukin-6, and tumor necrotic factor-α production. It also enhanced the neurite length of Neuro2a cells, which was linked to the upregulation of TuJ1 and MAP2 expressions. In conclusion, these findings indicate that the ethanolic extract of EO fruits has promising neuroprotective potential to exhibit antineuroinflammation activity and accelerative effect on neurite outgrowth in vitro. Therefore, EO fruit extract can be considered a novel herbal medicine candidate for managing neuroinflammatory diseases.

Human Hazard Assessment Using Drosophila Wing Spot Test as an Alternative In Vivo Model for Genotoxicity Testing-A Review.

Genomic instability, one of cancer's hallmarks, is induced by genotoxins from endogenous and exogenous sources, including reactive oxygen species (ROS), diet, and environmental pollutants. A sensitive in vivo genotoxicity test is required for the identification of human hazards to reduce the potential health risk. The somatic mutation and recombination test (SMART) or wing spot test is a genotoxicity assay involving Drosophila melanogaster (fruit fly) as a classical, alternative human model. This review describes the principle of the SMART assay in conjunction with its advantages and disadvantages and discusses applications of the assay covering all segments of health-related industries, including food, dietary supplements, drug industries, pesticides, and herbicides, as well as nanoparticles. Chemopreventive strategies are outlined as a global health trend for the anti-genotoxicity of interesting herbal extract compounds determined by SMART assay. The successful application of Drosophila for high-throughput screening of mutagens is also discussed as a future perspective.

Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions. AIM OF THE STUDY: The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety. MATERIALS AND METHODS: The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster. RESULTS: The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic. CONCLUSION: Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1ß, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.

Health-promoting bioactivity and in vivo genotoxicity evaluation of a hemiepiphyte fig, Ficus dubia.

Ficus species have been used as a typical component in food and folk medicine in Asia for centuries. However, little is known regarding the bioactivity and genotoxicity of the recently identified Ficus dubia (FD), an indigenous plant of the tropical evergreen rain forest. FD is unique from other Ficus species because of its highly sought-after red-brown latex. Antioxidant properties together with phenolic and flavonoid contents of FD were elucidated. Health-promoting characteristics were examined by studying the inhibition of enzymes as a drug target for diabetes, hypertension, Alzheimer's disease, and obesity, together with anticancer ability against human colorectal adenocarcinoma, human hepatocellular carcinoma, human ovarian carcinoma, human prostate adenocarcinoma, and human lung carcinoma. Besides, FD genotoxicity was tested using the Drosophila wing spot test. Results showed that both FD root and latex exhibited antioxidant activity due to the presence of phenolics and flavonoids, specifically caffeic acid and cyanidin. The ethanolic fraction of FD root demonstrated a potent antidiabetic mechanism underlying α-glucosidase inhibitory activity similar to acarbose. This fraction also suppressed lung and ovarian cancer growth, possibly by G1 and G2/M arrest, respectively. All tested fractions lacked mutagenicity in vivo. Results indicated that FD can be developed as novel antidiabetic compounds; however, its bioactive compounds should be further identified.

Composition and Acute Inflammatory Response from Tetraponera rufonigra Venom on RAW 264.7 Macrophage Cells.

Tetraponera rufonigra (Arboreal Bicoloured Ant) venom induces pain, inflammation, and anaphylaxis in people and has an increased incident in Southeast Asia regions. The bioactive components and mechanism of action of the ant venom are still limited. The aim of this research was to identify the protein composition and inflammatory process of the ant venom by using RAW 264.7 macrophage cells. The major venom proteins are composed of 5' nucleotidase, prolyl endopeptidase-like, aminopeptidase N, trypsin-3, venom protein, and phospholipase A2 (PLA2). The venom showed PLA2 activity and represented 0.46 µg of PLA2 bee venom equivalent/µg crude venom protein. The venom induced cytotoxic in a dose- and time-dependent manner with IC20 approximately at 4.01 µg/mL. The increased levels of COX-2 and PGE2 were observed after 1 h of treatment correlating with an upregulation of COX-2 expression. Moreover, the level of mPGES-1 expression was obviously increased after 12 h of venom induction. Hence, our results suggested that the induction of COX-2/mPGEs-1 pathway could be a direct pathway for the ant venom-induced inflammation.

Boesenbergia rotunda extract accelerates human keratinocyte proliferation through activating ERK1/2 and PI3K/Akt kinases.

Boesenbergia rotunda (BR) has long been used as tradition medicine. For its pharmacological effects on wound healing, previous studies in an animal model provided convincing results that the ethanolic extract from the rhizome of this plant can stimulate wound healing. However, the mechanism about how this plant promotes wound healing at the molecular level has not been elucidated. As a step towards the development of wound healing agents, our current study utilized a human keratinocyte cell line (HaCaT) as an in vitro model to define the potential molecular mechanisms of BR extract in enhancing wound-healing. Our HPLC results showed that BR extract contained kaempferol as one of its potential compounds. The extract strongly promoted wound healing of HaCaT cell monolayer. This effect was eventually defined to be regulated through the ability of BR extract to induce cell proliferation. At the signaling level, we discovered that BR extract rapidly activated ERK1/2 and Akt phosphorylation upon the addition of the extract. Additionally, our experiments where specific inhibitors of MEK (U0126) and PI3K (LY294002) were utilized verified that BR enhanced cell proliferation and wound healing through stimulating the MAPK and PI3K/Akt signal transduction pathways. Moreover, direct inhibition of keratinocyte DNA synthesis by mitomycin C (MMC) could completely block the proliferative effects of BR extract. Nevertheless, data from Transwell migration assay revealed that BR extract did not promote keratinocyte migration. Altogether, we provided more evidence that BR possesses its wound healing-promoting action through the activation of proliferation and survival pathways, and our study suggests that BR is an interesting candidate to be developed as a wound healing-promoting agent.

Inactivation of AKT/NF­κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin.

The high activation of protein kinase B (AKT)/nuclear factor­κB (NF­κB) signaling has often been associated with the induction of non­small cell lung cancer (NSCLC) cell survival and resistance to cisplatin, which is one of the most widely used chemotherapeutic drugs in the treatment of NSCLC. The inhibition of AKT/NF­κB can potentially be used as a molecular target for cancer therapy. Eurycomalactone (ECL), a quassinoid from Eurycoma longifolia Jack, has previously been revealed to exhibit strong cytotoxic activity against the human NSCLC A549 cell line, and can inhibit NF­κB activity in TNF­α­activated 293 cells stably transfected with an NF­κB luciferase reporter. The present study was the first to investigate whether ECL inhibits the activation of AKT/NF­κB signaling, induces apoptosis and enhances chemosensitivity to cisplatin in human NSCLC cells. The anticancer activity of ECL was evaluated in two NSCLC cell lines, A549 and Calu­1. ECL decreased the viability and colony formation ability of both cell lines by inducing cell cycle arrest and apoptosis through the activation of pro­apoptotic caspase­3 and poly (ADP­ribose) polymerase, as well as the reduction of anti­apoptotic proteins Bcl­xL and survivin. In addition, ECL treatment suppressed the levels of AKT (phospho Ser473) and NF­κB (phospho Ser536). Notably, ECL significantly enhanced cisplatin sensitivity in both assessed NSCLC cell lines. The combination treatment of cisplatin and ECL promoted cell apoptosis more effectively than cisplatin alone, as revealed by the increased cleaved caspase­3, but decreased Bcl­xL and survivin levels. Exposure to cisplatin alone induced the levels of phosphorylated­AKT and phosphorylated­NF­κB, whereas co­treatment with ECL inhibited the cisplatin­induced phosphorylation of AKT and NF­κB, leading to an increased sensitization effect on cisplatin­induced apoptosis. In conclusion, ECL exhibited an anticancer effect and sensitized NSCLC cells to cisplatin through the inactivation of AKT/NF­κB signaling. This finding provides a rationale for the combined use of chemotherapy drugs with ECL to improve their efficacy in NSCLC treatment.

Artocarpus lakoocha Extract Inhibits LPS-Induced Inflammatory Response in RAW 264.7 Macrophage Cells.

:Artocarpus lakoocha Roxb. (AL) has been known for its high content of stilbenoids, especially oxyresveratrol. AL has been used in Thai traditional medicine for centuries. However, the role of AL in regulating inflammation has not been elucidated. Here we investigated the molecular mechanisms underlying the anti-inflammation of AL ethanolic extract in RAW 264.7 murine macrophage cell line. The HPLC results revealed that this plant was rich in oxyresveratrol, and AL ethanolic extract exhibited anti-inflammatory properties. In particular, AL extract decreased lipopolysaccharide (LPS)-mediated production and secretion of cytokines and chemokine, including IL-6, TNF-α, and MCP-1. Consistently, the extract inhibited the production of nitric oxide (NO) in the supernatants of LPS-stimulated cells. Data from the immunofluorescence study showed that AL extract suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. Results from Western blot analysis further confirmed that AL extract strongly prevented the LPS-induced degradation of IκB which is normally required for the activation of NF-κB. The protein expression of iNOS and COX-2 in response to LPS stimulation was significantly decreased with the presence of AL extract. AL extract was found to play an anti-inflammatory role, in part through inhibiting LPS-induced activation of Akt. The extract had negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Specifically, incubation of cells with the extract for only 3 h demonstrated the rapid action of AL extract on inhibiting the phosphorylation of Akt, but not ERK1/2. Longer exposure (24 h) to AL extract was required to mildly reduce the phosphorylation of ERK1/2, p38, and JNK MAPKs. These results indicate that AL extract manipulates its anti-inflammatory effects mainly through blocking the PI3K/Akt and NF-κB signal transduction pathways. Collectively, we believe that AL could be a potential alternative agent for alleviating excessive inflammation in many inflammation-associated diseases.

Alpha-mangostin, an active compound in Garcinia mangostana, abrogates anoikis-resistance in human hepatocellular carcinoma cells.

Anoikis-resistance is a critical step in cancer progression, especially during the process of metastasis. During this phase, the cancer phenotype that causes cell survival in detachment conditions, drug resistance, and epithelial-to-mesenchymal transition (EMT) is altered. Inhibition of anoikis-resistance can potentially be the molecular target in cancer therapy. Alpha-mangostin, an active compound in Garcinia mangostana, has been reported for its cell-death induction and its chemosensitizing and anti-metastatic properties in many cancer cell types, such as ovarian cancer, lung cancer, and hepatocellular carcinoma. We, therefore, have investigated whether alpha-mangostin could sensitize anoikis in human hepatocellular carcinoma (HepG2). The established anoikis-resistant HepG2 displayed more aggressive malignant behaviors, including rapid proliferation, doxorubicin resistance, up-regulated anti-apoptotic protein levels, and EMT phenotype. Alpha-mangostin significantly sensitized anoikis in HepG2 through the inhibition of cell survival by induced caspase-9, caspase-8 and caspase-3 activities, increased pro-apoptotic protein (Bax, Bim, t-Bid) levels, and decreased anti-apoptotic protein (c-FLIP, Mcl-1) levels. Besides, alpha-mangostin significantly reduced cell re-adhesion and migration, matrix metalloproteinases-2 (MMP-2) and MMP-9 secretions, and EMT-involved protein (N-cadherin, αV, ß1 integrin, and vimentin) expressions. AKT and ERK signaling pathways were dramatically suppressed, which indicated that alpha-mangostin inhibited anoikis-resistance via the inhibition of these pathways in HepG2. These findings support the development of alpha-mangostin to be used in the treatment of anoikis-resistant liver cancer.

Modulation of P-glycoprotein by Stemona alkaloids in human multidrug resistance leukemic cells and structural relationships.

BACKGROUND: Multidrug resistance (MDR) is a major reason for the failure of chemotherapy in the treatment of cancer patients. P-gp over-expression in MDR cancer cells is a multifactorial phenomenon with biochemical resistance mechanisms. Stemofoline (STF), isolated from Stemona bukillii, has been reported to be an MDR reversing compound. PURPOSE: This study investigated whether other Stemona alkaloids that had been purified from Stemonaceae plants exerted MDR modulation activity. METHODS: MTT assay was performed to determine the MDR reversing property of the alkaloids. Modulation of P-gp function by these compounds was investigated using cell cycle analysis and P-gp fluorescent substrate accumulation assays. P-gp expression was determined by Western blot analysis. We preliminarily examined the safety of these compounds in normal human fibroblasts and human peripheral blood mononuclear cells (PBMCs) using the MTT assay, and in red blood cells (human and rat) through in vitro hemolysis assays. RESULTS: Three of the eight alkaloids tested, isostemofoline (ISTF), 11Z -didehydrostemofoline (11Z-DSTF) and 11E-didehydrostemofoline (11E-DSTF), enhanced the chemotherapeutic sensitivity of MDR leukemic K562/Adr cells, which overexpressed P-gp. The P-gp functional studies showed that these three alkaloids increased the accumulation of P-gp substrates, calcein-AM (C-AM) and rhodamine123 (Rho 123) in K562/Adr cells, while this effect was not seen in drug sensitive parental K562 cells. Whereas, the alkaloids did not alter P-gp expression as was determined by Western blotting analysis. CONCLUSION: The alkaloids reversed MDR via the inhibition of P-gp function. For pharmaceutical safety testing, the alkaloids were found to be not toxic to normal human fibroblasts and PBMCs. Moreover, the effective compounds did not induce hemolysis in either human or rat erythrocytes. These compounds may be introduced as potential candidate molecules for treating cancers exhibiting P-gp-mediated MDR.

Anti-inflammatory and anti-insulin resistance activities of aqueous extract from Anoectochilus burmannicus.

This study investigated biological activities including antioxidative stress, anti-inflammation, and anti-insulin resistance of Anoectochilus burmannicus aqueous extract (ABE). The results showed abilities of ABE to scavenging DPPH and ABTS free radicals in a dose-dependent manner. Besides, ABE significantly reduced nitric oxide (NO) production in the lipopolysaccharide (LPS)-treated RAW 264.7 via inhibition of mRNA and protein expressions of nitric oxide synthase (iNOS). The LPS-induced mRNA expressions of cyclooxygenase-2 (COX-2) and interleukin 1ß (IL-1ß) were suppressed by ABE. Moreover, ABE exerted anti-insulin resistance activity as it significantly improved the glucose uptake in tumor necrosis factor (TNF)-α treated 3T3-L1 adipocytes. In addition, ABE at the concentration of up to 200 µg/mL was not toxic to human peripheral blood mononuclear cells (PBMCs) and did not induce mutations. Finally, the results of our study suggest the potential use of A. burmannicus as anti-inflammatory, anti-insulin resistance agents, or food supplement for prevention of chronic diseases.

Kuguacin J isolated from bitter melon leaves modulates paclitaxel sensitivity in drug-resistant human ovarian cancer cells.

We previously reported the multidrug resistance-reversing ability of kuguacin J (KJ) in cervical cancer cells via the inhibition of P-glycoprotein (P-gp) function. This study investigated whether KJ could promote cisplatin- and paclitaxel (PTX)-induced cancer cell death in drug-resistance human ovarian cancer cells (SKOV3). Cytotoxicity testing showed that SKOV3 was more resistant to cisplatin and PTX compared to drug-sensitive human ovarian cancer cells (A2780). The cytotoxicity of PTX was significantly increased in SKOV3 cells when co-treated with KJ. We found that enhancement of PTX toxicity in the cells was not related to P-gp inhibition. To elucidate the mechanism by which KJ increases PTX sensitivity, the expression of cell death involving proteins was analyzed by Western blot analysis. The results showed that PTX treatment increased the level of an anti-apoptotic protein, survivin, which may be involved in drug resistance in SKOV3. The co-treatment with PTX and KJ dramatically decreased the level of survivin and markedly induced cleavage of PARP and caspase-3, which are apoptotic-induced molecules. These findings may support the use of KJ as an effective chemosensitizer in combination with conventional chemotherapy to promote PTX sensitization in ovarian cancer patients.

Anti-inflammatory effects of proanthocyanidin-rich red rice extract via suppression of MAPK, AP-1 and NF-κB pathways in Raw 264.7 macrophages.

BACKGROUND/OBJECTIVES: Several pharmacological properties of red rice extract have been reported including anti-oxidant, anti-tumor, and reduced cancer cell invasion. This study was conducted to evaluate the anti-inflammatory effects of red rice extract on the production of inflammatory mediators in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages. MATERIALS/METHODS: Pro-inflammatory cytokines including tumor necrosis factor-α and interleukin-6 were determined by ELISA and cyclooxygenase-2 and inducible nitric oxide synthase expression was evaluated using western blot analysis. In addition, the signaling pathway controlling the inflammatory cascade such as nuclear factor kappa B (NF-κB), activator proteins-1 (AP-1), and mitogen-activated protein kinase (MAPK) was determined. RESULTS: Our results showed that red rice polar extract fraction (RR-P), but not non-polar extract fraction, inhibited interleukin-6, tumor necrosis factor-α, and nitric oxide production in LPS-induced Raw 264.7 cells. RR-P also reduced the expression of inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, activation of AP-1 and NF-κB transcription factor in the nucleus was abrogated by RR-P. RR-P inhibited the phosphorylation of extracellular signaling-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 MAPK signaling responsible for the expression of inflammatory mediators in LPS-stimulated Raw 264.7 cells. Based on chemical analysis, high amounts of proanthocyanidin and catechins were detected in the RR-P fraction. However, only proanthocyanidin reduced NF-κB and AP-1 activation in LPS-activated Raw 264.7 cells. CONCLUSION: These observations suggest that the anti-inflammatory properties of RR-P may stem from the inhibition of pro-inflammatory mediators via suppression of the AP-1, NF-κB, and MAPKs pathways.

Chemosensitizing effects of synthetic curcumin analogs on human multi-drug resistance leukemic cells.

Curcumin analogs were synthesized and their multi-drug resistance (MDR) reversing properties were determined in human MDR leukemic (K562/Adr) cells. Four analogs, 1,7-bis-(3,4-dimethoxy-phenyl)-hepta-1,6-diene-3,5-dione (1J), 2,6-bis-(4-hydroxy-3-methoxy-benzylidene)-cyclohexanone (2A), 2,6-bis-(3,4-dihydroxy-benzylidene)-cyclohexanone (2F) and 2,6-bis-(3,4-dimethoxy-benzylidene)-cyclohexanone (2J) markedly increased the sensitivity of K562/Adr cells to paclitaxel (PTX) for 8-, 2-, 8- and 16- folds, respectively and vinblastine (Vin) for 5-, 3-, 12- and 30- folds, respectively. The accumulation of P-gp substrates, Calcein-AM, Rhodamine 123 and Doxorubicin, was significantly increased by 1J (up to 6-, 11- and 22- folds, respectively) and 2J (up to 7-, 12- and 17- folds, respectively). Besides 2A, 2F and 2J dramatically decreased P-gp expression in K562/Adr cells. These results could be summarized in the following way. Analog 1J inhibited only P-gp function, while 2A and 2F inhibited only P-gp expression. Interestingly, 2J exerts inhibition of both P-gp function and expression. The combination index (CI) of combination between 2J and PTX (0.09) or Vin (0.06) in K562/Adr cells indicated strong synergistic effects, which likely due to its MDR reversing activity. Moreover, these analogs showed less cytotoxicity to peripheral mononuclear cells (human) and red blood cells (human and rat) suggesting the safety of analogs for further animal and clinical studies.