Gynura procumbens (Lour.) Merr. extract attenuates monocyte adherence to endothelial cells through suppression of the NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Gynura procumbens (Lour.) Merr. (GP) is a herbaceous plant that grows in Malaysia and other parts of Southeast Asia. The herb is consumed as a remedy for various inflammatory-associated diseases, such as cancer, rheumatism, hypertension, diabetes mellitus and hyperlipidemia. Scientific studies demonstrate that GP extract possesses cardioprotective and anti-inflammatory effects. Cardiovascular disease is mainly caused by atherosclerosis, and inflammation plays a major role in all phases of atherosclerosis. The early inflammatory events in atherogenesis are the activation of endothelial cells and the recruitment of monocytes. AIM OF THE STUDY: This study aimed to evaluate the inhibitory effect of 80% ethanol extract of GP leaves (GPE) on the adherence of monocytes to the activated human endothelial cells and its underlying mechanism. MATERIAL AND METHODS: Qualitative and quantitative analyses of the extract were carried out by using a validated HPLC and UHPLC-MS/MS methods. The MTT test was used to select the range of concentration of extract for this study. The effect of GPE on TNF-α-induced monocyte-endothelial interaction was determined by the in vitro adhesion assay. Expression of cell surface proteins (ICAM-1, VCAM-1) and phosphorylation of nuclear factor kappa B (NF-κB) were determined by western blot, while expression of a chemokine (MCP-1) was identified by an enzyme-linked immunosorbent assay. RESULTS: HPLC and UHPLC-MS/MS analyses indicated that GPE contained chlorogenic acid, nicotiflorin and astragalin as the major compounds. GPE at 20, 40 and 60 µg/mL concentrations showed a significant reduction in monocyte adherence to endothelial cells and expression of ICAM-1 and MCP-1. However, only GPE at concentrations of 40 and 60 µg/mL was able to reduce VCAM-1 expression. Furthermore, GPE significantly inhibited IKKα/ß, IκBα, NF-κB phosphorylation and NF-κB translocation. CONCLUSION: In conclusion, GPE may inhibit monocyte adherence to the activated endothelial cells and expression of ICAM-1, VCAM-1 and MCP-1, which are important proteins for monocyte-endothelial interaction, by suppressing the NF-κB signaling pathway. The results of this study support the traditional use of GPE to counteract inflammation-associated diseases and suggest that GP can be a potential source for bioactive compounds for the development of anti-inflammatory agents to prevent atherosclerosis.

Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages.

Triple-negative breast cancer is the main type of breast carcinoma that causes mortality among women because of the limited treatment options and high recurrence. Chronic inflammation has been linked with the tumor microenvironment (TME) in breast cancer progression. Clinacanthus nutans (CN) has gained much attention because of its anticancer properties, but its mechanism remains unclear. We aimed to study the qualitative phytochemical content and elucidate the cytotoxicity effects of CN on human triple-negative breast cancer (TNBC), MDA-MB-231 and human macrophage-like cells such as THP-1 by using sulforhodamine B (SRB) assay. As highly metastatic cells, MDA-MB-231 cells can migrate to the distal position, the effect of CN on migration were also elucidated using the scratch assay. The CN effects on ameliorating chronic inflammation in TME were studied following the co-culture of MDA-MB-231/THP-1 macrophages. The cytokine expression levels of IL-6, IL-1ß and tumor necrosis factor-alpha (TNF-α) were determined using ELISA assays. The results showed that both ethanolic and aqueous CN extracts contained alkaloid, phenol and tannin, flavonoid, terpenoid, glycoside and steroid. However, saponin was only found in the aqueous extract of CN. CN was not cytotoxic to both MDA-MB-231 and THP-1 cells. The ability of MDA-MB-231 to migrate was also not halted by CN treatment. However, CN ethanol extract decreased IL-6 at 25 µg/mL (p = 0.02) and 100 µg/mL (p = 0.03) but CN aqueous extract increased IL-6 expression at 50 µg/mL (p = 0.08) and 100 µg/mL (p = 0.02). IL-1ß showed decreased expression after treated with CN ethanol and CN aqueous both at 25 µg/mL (p = 0.03). TNF-α were significantly decreased after CN ethanol treatment at concentration 25- (p = 0.001), 50- (p = 0.000) and 100 µg/mL (p = 0.000). CN aqueous extract slightly inhibited TNF-α at all 25-50- and 100 µg/mL (p = 0.001, p = 0.000, p = 0.000, respectively). Overall, CN acts by ameliorating the pro-inflammatory condition in the TME and may be a potential strategy for its anticancer mechanism on highly metastatic breast cancer condition. The major pathways that link both cancer and inflammation were NF-κB and STATs thus further study on the upstream and downstream pathways is needed to fully understand the mechanism of CN extracts in cooling the inflamed TME in breast cancer.

Antimutagenic, Cytoprotective and Antioxidant Properties of Ficus deltoidea Aqueous Extract In Vitro.

Ficus deltoidea var. deltoidea is used as traditional medicine for diabetes, inflammation, and nociception. However, the antimutagenic potential and cytoprotective effects of this plant remain unknown. In this study, the mutagenic and antimutagenic activities of F. deltoidea aqueous extract (FDD) on both Salmonella typhimurium TA 98 and TA 100 strains were assessed using Salmonella mutagenicity assay (Ames test). Then, the cytoprotective potential of FDD on menadione-induced oxidative stress was determined in a V79 mouse lung fibroblast cell line. The ferric-reducing antioxidant power (FRAP) assay was conducted to evaluate FDD antioxidant capacity. Results showed that FDD (up to 50 mg/mL) did not exhibit a mutagenic effect on either TA 98 or TA 100 strains. Notably, FDD decreased the revertant colony count induced by 2-aminoanthracene in both strains in the presence of metabolic activation (p < 0.05). Additionally, pretreatment of FDD (50 and 100 µg/mL) demonstrated remarkable protection against menadione-induced oxidative stress in V79 cells significantly by decreasing superoxide anion level (p < 0.05). FDD at all concentrations tested (12.5-100 µg/mL) exhibited antioxidant power, suggesting the cytoprotective effect of FDD could be partly attributed to its antioxidant properties. This report highlights that F. deltoidea may provide a chemopreventive effect on mutagenic and oxidative stress inducers.

Zinc L-Carnosine Protects CCD-18co Cells from L-Buthionine Sulfoximine-Induced Oxidative Stress via the Induction of Metallothionein and Superoxide Dismutase 1 Expression.

Zinc L-carnosine (ZnC) is the chelate form of zinc and L-carnosine and is one of the zinc supplements available in the market. This study aims to determine the protective effects of ZnC against L-buthionine sulfoximine (BSO)-induced oxidative stress in CCD-18co human normal colon fibroblast cell line. CCD-18co cells were pretreated with ZnC (0-100 µM) for 24 h before the induction of oxidative stress by BSO (1 mM) for another 24 h. Results from this present study demonstrated that ZnC up to the concentration of 100 µM was not cytotoxic to CCD-18co cells. Induction with BSO significantly increased the intracellular reactive oxygen species (ROS) levels and reduced the intracellular glutathione (GSH) levels in CCD-18co cells. Pretreatment with ZnC was able to attenuate the increment in intracellular ROS level in CCD-18co cells significantly in a concentration-dependent manner. However, ZnC did not have any effects on intracellular GSH levels and Nrf2 activation. Mechanistically, pretreatment with ZnC was able to upregulate the expression of metallothionein (MT) and superoxide dismutase 1 (SOD1) in CCD-18co cells. Results from dual-luciferase reporter gene assay reported that ZnC was able to increase the MRE-mediated relative luciferase activities in a concentration-dependent manner, suggesting that the induction of MT expression by ZnC was due to the activation of MTF-1 signaling pathway. Taken together, our current findings suggest that ZnC can protect CCD-18co cells from BSO-induced oxidative stress via the induction of MT and SOD1 expression.

Dendritic cells pulsed with generated tumor cell lysate from Phyllanthus amarus Schum. & Thonn. induces anti-tumor immune response.

BACKGROUND: Dendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. In the present study, 80% ethanol extract of Phyllanthus amarus was used to generate tumor lysate (TLY) derived from HCT 116 and MCF-7 cancer cell lines via induction of apoptosis. Monocyte-derived DCs were generated ex vivo from the adherent population of peripheral blood mononuclear cells (PBMCs). The generated TLY were used to impulse DCs to investigate its effect on their cellular immune functions including antigen presentation capacity, phagocytic activity, chemotaxis capacity, T-cell proliferation and cytokines release. METHODS: The effect of P. amarus-generated TLY on DCs maturation was evaluated by determination of MHC class I, II and CD 11c expression as well as the co-stimulatory molecules CD 83 and 86 by using flow cytometry. The phagocytic capacity of TLY-pulsed DCs was investigated through FITC-dextran uptake by using flow cytometry. The effect on the cytokines release including IL-12, IL-6 and IL-10 was elucidated by using ELISA. The migration capacity and T cell proliferation activity of pulsed DCs were measured. The relative gene expression levels of cytokines were determined by using qRT-PCR. The major constituents of P. amarus extract were qualitatively and quantitatively analyzed by using validated reversed-phase high performance liquid chromatography (HPLC) methods. RESULTS: P. amarus-generated TLY significantly up-regulated the expression levels of MHC class I, CD 11 c, CD 83 and 86 in pulsed DCs. The release of interleukin IL-12 and IL-6 was enhanced by TLY-DCs at a ratio of 1 DC: 3 tumor apoptotic bodies (APO), however, the release of IL-10 was suppressed. The migration ability as well as allogeneic T-cell proliferation activities of loaded DCs were significantly enhanced, but their phagocytic capacity was highly attenuated. The gene expression profiles for IL-12 and IL-6 of DCs showed increase in their mRNA gene expression in TLY pulsed DCs versus unloaded and LPS-treated only DCs. CONCLUSION: The effect of P. amarus-generated TLY on the immune effector mechanisms of DCs verified its potential to induce an in vitro anti-tumor immune response against the recognized tumor antigen.

Water extract of Clinacanthus nutans leaves exhibits in vitro, ex vivo and in vivo anti-angiogenic activities in endothelial cell via suppression of cell proliferation.

BACKGROUND: Clinacanthus nutans (Burm. f.) Lindau. has traditionally been using in South East Asia countries to manage cancer. However, scientific evidence is generally lacking to support this traditional claim. This study aims to investigate the in vitro, ex-vivo and in vivo effects of C. nutans extracts on angiogenesis. METHODS: C. nutans leaves was extracted with 50-100% ethanol or deionised water at 1% (w/v). Human umbilical veins endothelial cell (HUVEC) proliferation was examined using MTT assay. The in vitro anti-angiogenic effects of C. nutans were assessed using wound scratch, tube formation and transwell migration assays. The VEGF levels secreted by human oral squamous cell carcinoma (HSC-4) cell and HUVEC permeability were also measured. Besides, the rat aortic ring and chick embryo chorioallantoic membrane (CAM) assays, representing ex vivo and in vivo models, respectively, were performed. RESULTS: The MTT assay revealed that water extract of C. nutans leaves exhibited the highest activity, compared to the ethanol extracts. Therefore, the water extract was chosen for subsequent experiments. C. nutans leaf extract significantly suppressed endothelial cell proliferation and migration in both absence and presence of VEGF. However, the water extract failed to suppress HUVEC transmigration, differentiation and permeability. C. nutans water extract also did not suppress HSC-4 cell-induced VEGF production. Importantly, C. nutans water extract significantly abolished the sprouting of vessels in aortic rings as well as in chick embryo CAM. CONCLUSION: In conclusion, these findings reveal potential anti-angiogenic effects of C. nutans, providing new evidence for its potential application as an anti-angiogenic agent.

Inhibitory effects of compounds from Phyllanthus amarus on nitric oxide production, lymphocyte proliferation, and cytokine release from phagocytes.

Standardized extract of Phyllanthus amarus has previously been shown to have a strong inhibitory effect on phagocytic activity of human neutrophils. The current study was carried out to evaluate the effects of constituents of the extract of P. amarus on nitric oxide (NO) production as well as lymphocyte proliferation and cytokine release from phagocytes. Three compounds, ethyl 8-hydroxy-8-methyl-tridecanoate, 7ß,19α dihydroxy-urs-12-ene, and 1,7,8-trihydroxy-2-naphtaldehyde, together with seven known compounds were isolated from the whole plant of P. amarus. The isolated compounds and reference standards, ie, gallic acid, ellagic acid, corilagin, and geraniin, which were quantitatively analyzed in the extracts, were evaluated for their effects on immune cells. Among the compounds tested, the lignans, especially phyltetralin and phyllanthin, showed strong inhibition on lymphocyte proliferation with half maximal inhibitory concentration (IC50) values of 1.07 µM and 1.82 µM, respectively. Ethyl 8-hydroxy-8-methyl-tridecanoate and 1,7,8-trihydroxy-2-naphtaldehyde exhibited strong inhibition on nitric oxide production with IC50 values of 0.91 µM and 1.07 µM, respectively. Of all the compounds, corilagin was the strongest inhibitor of tumor necrosis factor-α release with an IC50 value of 7.39 µM, whereas geraniin depicted the strongest inhibitory activity on interleukin-1ß release with an IC50 value of 16.41 µM. The compounds constituting the extract of P. amarus were able to inhibit the innate immune response of phagocytes at different steps.

Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage.

The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action.

Assessment of cytotoxicity and genotoxicity of stem bark extracts from Canarium odontophyllum Miq. (dabai) against HCT 116 human colorectal cancer cell line.

BACKGROUND: Canarium odontophyllum Miq. is a plant species widely known as 'dabai' and can be vastly found in Sarawak. The aim of this study was to assess the cytotoxic and genotoxic effect of extracts from stem bark of C. odontophyllum against HCT 116 human colorectal cancer cell line. METHOD: The IC50 values of the aqueous, methanol, and acetone extracts against HCT 116 cells as well as the acetone extract against human colon fibroblast cell line CCD-18co were determined using the MTT assay. The concentration of the extracts ranged from 12.5 to 200 µg/ml at treatment time of 24, 48 and 72 h. Annexin V-FITC/PI labelling assay was employed to determine mode of HCT 116 cell death induced by acetone extract at 48 h. The DNA damage induced by the extract in HCT 116 cells was detected using alkaline comet assay at 30 min of IC10 and IC25 treatment. RESULTS: Acetone extract exhibited the highest cytotoxic effect against HCT 116 cells compared to methanol and aqueous extract at 24, 48 and 72 h. Despite no cytotoxic effect by acetone extract against CCD-18co cells at 24 and 48 h, however at 72 h, CCD-18co cells proliferated. Apoptosis assessment using Annexin V-FITC/PI labelling assay revealed that the primary cell death was via apoptosis after 48 h treatment. Low doses of acetone extract from stem bark of C. odontophyllum showed significant DNA damage in HCT 116 cells with tail moment of 6.187 ± 0.718 A.U and 7.877 ± 0.142 A.U, respectively. CONCLUSIONS: Acetone extract from stem bark of C. odontophyllum has high potential in the development of anticancer agent against HCT 116 cells with no cytotoxic effect against human colon fibroblast cells.