Extract-treated Cells Research Articles

Validation of target proteins of down-regulated miR-221-5p in HeLa cells treated with Polyalthia longifolia leaf extract using label-free quantitative proteomics approaches.

The current study was conducted to validate the target proteins of down-regulated miR-221-5p in HeLa cells treated with P. longifolia leaf extract. The validation was done by label-free quantitative proteomics approaches, Gene Ontology (GO) and protein-protein interaction analyses after the cells transfected with miRNA mimics or miRNA inhibitor. The LC-ESI-MS/MS identified a total of 1061, 668, 564 and 940 proteins from untransfected and untreated HeLa cells, untransfected P. longifolia leaf extract-treated HeLa cells, miR-221-5p mimic-transfected P. longifolia leaf extract-treated HeLa cells and anti-miR-221-5p-transfected P. longifolia leaf extract-treated HeLa cells, respectively. The proteomic, GO and protein-protein interaction analyses showed that P. longifolia treatment regulated various protein expressions in HeLa cells, namely tropomyosin, PRKC apoptosis WT1 regulator protein (PAWR), alpha-enolase and beta-enolase, which induced apoptotic cell death after the down-regulation of miR-221-5p. Conclusively, this study showed P. longifolia leaf extract's vital contribution in regulating various protein expressions in HeLa cervical cancer cells to induce apoptotic cell death after downregulation miR-221-5p.

LEAVES OF ANDROGRAPHIS PANICULATA IS AN ANTIOXIDANT AND ANTICANCER AGENT

Objective: Andrographis paniculata (Family: Acanthaceae) is a well-known medicinal plant used in the Indian traditional system of medicine for the treatment of many chronic diseases. The present study was aimed to quantify secondary metabolites, determine antioxidant, and anticancer activity of ethanol extract of A. paniculata leaves.
 Methods: Leaf sample was macerated with ethanol solvent. Alkaloids, terpenoids, saponins, phenols, and flavonoids were quantified with standard calibrations. The antioxidant potential was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) assays. In vitro anticancer activity was evaluated using human epithelial type 2 (HEp-2) cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to estimate the cytotoxicity of the extracts. Apoptotic and necrotic effects were characterized by DNA fragmentation assay and fluorescence microscopy using the dual acridine orange/ethidium bromide (AO/EB) staining method.
 Results: The phytochemical analysis reveals the presence of alkaloids, saponins, phenols, flavonoids, terpenoids, and steroids. Alkaloids, terpenoids, saponins, phenol, and flavonoid content were recorded as follows: 9.84%, 8.42%, 13.94%, 44.37 mg gallic acid equivalent/100 g, and 904 mg quercetin equivalent/100 g, respectively. The antioxidant activity from DPPH, ABTS, and FRAP assays showed dose-dependent inhibition of free radicals. In cell viability tests, cell death with increasing extract concentration was observed. DNA fragmentation and AO/EB stain confirmed apoptosis and necrosis in extract-treated cells.
 Conclusion: The results indicate that A. paniculata is a promising source for the development of antioxidant and anticancer drugs.

Apoptosis induction of crude water soluble Momordica charantia (chinese and indian bitter gourd) in human lung cancer cell and zebrafish embryo

A therapy which can target apoptosis will be effective for cancer treatment. This preliminary study attempted in vitro and in vivo to target apoptosis capability of Momordica charantia extracts. Here, we describe that the crude hot and cold extraction of two varieties (Chinese and Indian) of M.charantia, induced apoptosis in human lung cancer cell line A549. This was obtained from the cell viability assay where crude extracts were incubated for 24 hours and tested with 3-(4,5-Dimethylthiazol-2-yl)-2,5 Diphenyltetrazolium Bromide (MTT) assay. The inhibitory concentration, IC50 was obtained from the cell viability graph and further used in the upstream assays. The apoptotic morphology was presented by the crude extract-treated cells with a comparison to a positive control cisplatin. The percentage of apoptosis was obtained from a flow cytometric analysis using Hoechst 33342. Induction of apoptosis in vivo was tested using the extracts on dechorionated zebrafish embryo for 24 hours and monitored up to 72-hour post fertilization. Induction of apoptosis in A549 by crude water extracts revealed a decrease in cell viability giving a low IC50. The IC50 of cisplatin was 17.3 µg/ml, Chinese Hot Aqueous was 32.5 µg/ml, Chinese Cold Aqueous was 28.1 µg/ml, Indian Hot Aqueous was 36.9 µg/ml and Indian Cold Aqueous was 26.7 µg/ml significant to the control.The initiation of apoptosis by the crude water extracts was also evidence in vivo in zebrafish embryos with results demonstrating significant changes morphologically. There is a potential for the Indian Cold Aqueous to act as a chemo preventive agent as it has the overall best IC50, percentage of apoptotic cells and changes in the morphology of cell and the zebrafish embryo.

Epithelial-Mesenchymal Transition in Asthma Airway Remodeling Is Regulated by the IL-33/CD146 Axis.

Epithelial-mesenchymal transition (EMT) is essential in asthma airway remodeling. IL-33 from epithelial cells is involved in pulmonary fibrosis. CD146 has been extensively explored in cancer-associated EMT. Whether IL-33 regulates CD146 in the EMT process associated with asthma airway remodeling is still largely unknown. We hypothesized that EMT in airway remodeling was regulated by the IL-33/CD146 axis. House dust mite (HDM) extract increased the expression of IL-33 and CD146 in epithelial cells. Increased expression of CD146 in HDM-treated epithelial cells could be blocked with an ST2-neutralizing antibody. Moreover, HDM-induced EMT was dependent on the CD146 and TGF-β/SMAD-3 signaling pathways. IL-33 deficiency decreased CD146 expression and alleviated asthma severity. Similarly, CD146 deficiency mitigated EMT and airway remodeling in a murine model of chronic allergic airway inflammation. Furthermore, CD146 expression was significantly elevated in asthma patients. We concluded that IL-33 from HDM extract-treated alveolar epithelial cells stimulated CD146 expression, promoting EMT in airway remodeling in chronic allergic inflammation.

The effects of microcystin-LR in Oryza sativa root cells: F-actin as a new target of cyanobacterial toxicity.

Microcystins are toxins produced by cyanobacteria, notorious for negatively affecting a wide range of living organisms, among which several plant species. Although microtubules are a well-established target of microcystin toxicity, its effect on filamentous actin (F-actin) in plant cells has not yet been studied. Τhe effects of microcystin-LR (MC-LR) and an extract of a microcystin-producing freshwater cyanobacterial strain (Microcystis flos-aquae TAU-MAC 1510) on the cytoskeleton (F-actin and microtubules) of Oryza sativa (rice) root cells were studied with light, confocal, and transmission electron microscopy. Considering the role of F-actin in endomembrane system distribution, the endoplasmic reticulum and the Golgi apparatus in extract-treated cells were also examined. F-actin in both MC-LR- and extract-treated meristematic and differentiating root cells exhibited time-dependent alterations, ranging from disorientation and bundling to the formation of ring-like structures, eventually resulting in a collapse of the F-actin network after longer treatments. Disorganization and eventual depolymerization of microtubules, as well as abnormal chromatin condensation were observed following treatment with the extract, effects which could be attributed to microcystins and other bioactive compounds. Moreover, cell cycle progression was inhibited in extract-treated roots, specifically affecting the mitotic events. As a consequence of F-actin network disorganization, endoplasmic reticulum elements appeared stacked and diminished, while Golgi dictyosomes appeared aggregated. These results support that F-actin is a prominent target of MC-LR, both in pure form and as an extract ingredient. Endomembrane system alterations can also be attributed to the effects of cyanobacterial bioactive compounds (including microcystins) on the F-actin cytoskeleton.

A protective polymorphism in MMP16, improved blood gas levels, and chronic obstructive pulmonary diseases: Family and two population-based studies.

The aberrant expression of matrix metalloproteinases (MMPs) is known to contribute to the pathogenesis of airway remodeling and alveolar disruption in chronic obstructive pulmonary disease (COPD). In the discovery stage, 11 COPD from five families were subjected to whole-genome sequencing, and 21 common polymorphisms in MMPs and TIMPs were identified. These polymorphisms were genotyped in two subsequent verification studies. Of these polymorphisms, c.2392G>A (rs2664370T>C) and c.4158C>A (rs2664369T>G) in MMP16 remained significantly different. Functionally, we found that MMP16 expression was significantly increased in peripheral blood monocytes (PBMCs) from COPD and in cigarette smoke extract-treated 16HBE cells compared with controls. This was also shown by bioinformatics analysis. COPD carrying rs2664370CC showed decreased levels of MMP16 in the plasma and in PBMCs compared with those carrying CT and TT. Treatment with hsa-miR-576-5p mimics led to a greater reduction in luciferase reporter activity in cells transfected with rs2664370CC. Moreover, blood levels of base excess, PCO2 , and PO2 in COPD with rs2664370CC were significantly lower than those with rs2664370CT+TT. Taken together, these results demonstrate that the rs2664370T>C polymorphism in MMP16 protects against the risk of COPD, likely by favoring interaction with hsa-miR-576-5p, leading to reduced MMP16 expression and improved blood gas levels.

Phenolic Extract of Seagrass, Halophila ovalis Activates Intrinsic Pathway of Apoptosis in Human Breast Cancer (MCF-7) Cells

The marine ecosystem is considered as a treasure of numerous novel biologically active molecules. We investigated the anticancer potential of the phenolic extract of Halophila ovalis in breast cancer (MCF-7) cells and characterized the possible underlying molecular mechanism. The phenolic extract (5 µl) of H. ovalis effectively inhibited the growth of MCF-7 cells. The results of DAPI staining indicated that this phenolic extract potently induces apoptosis in MCF-7 cells which was observed by increased chromatin condensation in the treated cells. An increased expression of the active fragments of an executioner caspase, caspase 3 in phenolic extract-treated MCF-7 cells further confirms this apoptosis induction. In consequence, the loss of mitochondrial membrane potential was noticed in treated cells. The protein expression analyzes show decreased expression of the anti-apoptotic protein, Bcl-2, and DNA repair enzyme, PARP in treated cells indicating the probable molecular targets of apoptosis. Further, the phenolic extract of H. ovalis blocked the antioxidant defense system in MCF-7 cells by down-regulating the protein expression of a major transcription factor, Nrf-2 and regulatory antioxidant enzymes, SOD-2 and HO-1. These results show the presence of chemopreventive compound(s) in the phenolic extract, which offers a platform for future studies to identify the active principles.

Xao tam phan (Paramignya trimera) methanol extract induced apoptosis in hepatocellular carcinoma HepG2 cell line in vitro

Introduction: Xao Tam Phan (Paramignya trimera) has long been used in Viet Nam as an herbal medicine for the treatment of Hepatitis, hepatocellular carcinoma, and diabetes. This study aimed to determine the anti-proliferation effect of Paramignya trimera extract (P. trimera extract) on HepG2 hepatocellular carcinoma cells.
 Methods: AlamarBlue assay was used to determine the IC50 values of P. trimera extract on HepG2 cells. Adipose-derived stem cells (ADSCs) was used as normal cell control. For apoptosis examination, P. trimera extract-treated HepG2 cells were incubated with Annexin V/Propidium iodide (PI). Then they have been analyzed their expression of Annexin-V and PI by flow cytometry. The cell nuclear degradation also was evaluated by PI/Hoechst 33342 staining assay.
 Results: Doxorubicin and P. trimera extract IC50 values on HepG2 cells were 55.13 +/- 2.028 ng/ml and 582.533 +/- 16.521 mg/ml, respectively. Those on ADSCs were 5.96 +/- 0.56 ng/ml and 268.976 +/- 19.325 mg/ml, respectively. Side effect index value (SEI) of P. trimera extract was 2.175 +/- 0.12, and the SEI of doxorubicin was 8.71 +/- 0.36. Flow cytometry analysis indicated significant apoptosis on P. trimera extract-treated HepG2 cells at a dose of 500 mg/ml (32.39 +/- 2.28% apoptotic cells, and 14.63 +/- 1.59% necrotic cells). Nuclear aggregation and degradation was seen on 500 mg/ml P. trimera treated HepG2 cells.
 Conclusion: P. trimera extract could inhibit HepG2 hepatocellular carcinoma cell proliferation by inducing apoptosis.

GC-MS analysis of Myrtus communis extract and its antibacterial activity against Gram-positive bacteria

BackgroundMyrtus communis is a typical plant of Mediterranean area. The different parts of this plant such as berries, branches, and leaves have been used worldwide as a traditional/folk medicine for the treatment of various ailments and diseases.MethodsEthanolic leaf extract of the plant was prepared by Soxhlet extraction method. Zone of inhibition, minimum inhibitory concentration and minimal bactericidal concentration were determined by well diffusion method and microplate alamar blue assay. GC-MS analysis was carried out to identify the compounds present in the extract. Microscopy and ImageJ software were used respectively for morphology and cell-length measurements. GraphPad Prism was used for statistical analysis.ResultsThe ethanolic extract showed strong inhibitory effect against Gram-positive and acid-fast bacteria with significant inhibition-zone size (9–25 mm), MIC (4.87–78 μg/ml), as well as MBC (0.3–20 mg/ml). However, no effect was observed on the growth of Gram-negative bacteria. The growth inhibition was found to be associated with the damage of cell wall as the extract-treated cells were sensitive to cell wall-targeting antibiotics and displayed the cell wall damage-depicting morphological defects. GC-MS analysis confirmed the presence of novel compounds in addition to the most representative compounds of the essential oils/extracts of M. communis of other country origins.ConclusionThese results demonstrate that M. communis leaf extract could be the source of compounds to be used for the treatment of Gram-positive bacterial infections. This is the first report, which provides insights into the mechanism of action of the extract in inhibiting the growth of Gram-positive bacteria.

Origanum vulgare extract induces apoptosis in Molt-4 leukemic cell line

OBJECTIVE: The purpose of this paper is to investigate the effect of Origanum vulgare extract as a plant with high anti-oxidant components on the induction of cell death in the Molt-4 cell line. BACKGROUND: Acute lymphocytic leukemia is characterized by the accumulation of a large number of lymphoblastic cells with high oxidant levels. METHODS: MTT assay was performed to determine the effect of O.vulgare extract on Molt-4 cells viability and the amount of 50% inhibitory concentration (IC50) was calculated. Changes in the expression of BAX and BCL-2 genes as involved in apoptosis and Nrf2 gene as a transcription factor of anti-oxidant genes in O.vulgare extract-treated Molt-4 cells were measured with Real-Time PCR. Treated Molt-4 cells were used to determine the stages of early and late apoptosis, and necrosis using acridine orange/ethidium bromide double staining. RESULTS: The results suggest survival inhibition and induction of apoptosis in Molt-4 cells treated with O.vulgare extract. Against Bax and Nrf2 genes expression, the expression of Bcl-2 gene has been reduced in Molt-4 cells following1/5 IC50 concentration of O. vulgare extract treatment. CONCLUSION: Given the oxidant drugs used in ALL treatment, and increased levels of oxidative stress in leukemic cells, induction of apoptosis by an anti-oxidant plant extract seems to be a promising way in leukemia treatment.

Methanolic Bark Extract of Abroma augusta (L.) Induces Apoptosis in EAC Cells through Altered Expression of Apoptosis Regulatory Genes.

Abroma augusta (L.), one of the herbal medicinal plants, is widely used for treatment of various maladies. The present study was initiated to determine the antioxidant, hemolytic, cytotoxicity, and anticancer activities of methanolic extract from the bark of the plant. The phytochemical screening was done by analyzing different phytochemicals present in the extract. We observed the presence of alkaloids, steroids, terpenoids, flavonoids, reducing sugars, and glycosides in the bark extract which showed the highest antioxidant capacity. Antioxidant potential of the methanolic extract was evaluated in vitro by DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay method. This extract showed prominent scavenging activity with IC50 value of 38.65 μg/ml. The hemolytic activity of the extract was evaluated at concentrations ranging from 250 to 1000 μg/ml. It was observed that the extract induced hemolysis percentage of 9.41% to 4.1%, which implies that the extract has no potent hemolytic activity. Cytotoxicity and anticancer activities were observed on Ehrlich ascites carcinoma (EAC) cells. In addition, the bark showed promising cytotoxicity with IC50 value of 329.41 μg/ml, and the study indicated that the extract was capable of inhibiting EAC cell growth by 75.5% when administered at 100 mg/kg/day body weight intraperitoneally for five consecutive days to Swiss albino mice. Morphological change of apoptotic cell was determined by fluorescence and optical microscopy. DNA fragmentation is another marker for apoptosis, and the bark extract-treated EAC cells showed smeared and fragmented DNA bands. Apoptosis correlated well with the upregulation of p53 and Bax and also with the downregulation of NF-κB and Bcl-2. Furthermore, activity and interaction of two A. augusta compounds were tested through molecular docking simulation study. In conclusion, our results suggest that A. augusta bark has the potential to be considered as an anticancer agent.

Fridericia platyphylla (Cham.) L.G. Lohmann root extract exerts cytotoxic and antiproliferative effects on gastric tumor cells and downregulates BCL-XL, BIRC5, and MET genes.

Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL-1 and 69.38, 41.73, and 52.39 µg mL-1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL-1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL-1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL-1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated.

Bulbine frutescens phytochemical inhibits notch signaling pathway and induces apoptosis in triple negative and luminal breast cancer cells

Breast cancer (BCa) is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to BCa recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Bulbine frutescens (Asphodelaceae), a traditional medicinal plant in South Africa possess bioactive flavonoids and terpenoids. Polar (methanol) and non-polar (hexane) B. frutescens plant extracts were prepared. GC–MS analysis revealed the differential presence of secondary metabolites in both methanolic and hexane extracts. We hereby first time evaluated the anticancer potential of B. frutescens methanolic and hexane extract in triple-negative and luminal BCa cells. B. frutescens extracts significantly decreased cell viability (IC50 4.8–28.4 μg/ml) and induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells as confirmed by spectrophotometry and flow cytometry technique. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in extract-treated BCa cells. Fluorescence spectrophotometry and confocal microscopy showed B. frutescens induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells. Flow cytometric apoptosis analysis of B. frutescens extracts treated MDA-MB-231 cells showed ≈13% increase in early apoptotic population in comparison to non-treated cells. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens extracts. Moreover, RTPCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract-treated BCa cells. Western Blot analysis showed increased procaspase 3 protein expression in extract-treated BCa cells. In all the assays methanolic extract showed better anti-cancer properties. Literature-based identification of methanol soluble phytochemicals in B. frutescens and in silico docking study revealed Bulbineloneside D as a potent ϒ-secretase enzyme inhibitor. In comparison to standard notch inhibitor, lead phytochemical showed two additional hydrophobic interactions with Ala80 and Leu81 amino acids. In conclusion, B. frutescens phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. B. frutescens phytochemicals have the ability to downregulate the notch signaling pathway in triple-negative and luminal breast cancer cells.

In vitro apoptosis induction ability of methanolic extract of Paramignya trimera root (Xao tam phan) in breast cancer stem cells

Objective: Cancer has been considered as one of the world's leading causes of death. Recently, the Paramignya trimera plant, locally called ``Xao Tam Phan'', has become a popular Vietnamese medicinal herb that is used as alternative medicine for cancer treatment support with minimal side effects. In this study, we aimed to demonstrate the cytotoxicity of methanolic extract of Paramignya trimera on a Vietnamese breast cancer stem cell line (VNBRCA1) in vitro.
 Methods: We used the MTT assay to determine the cytotoxicity of the extract on VNBRCA1 cells and human fibroblast (HF) cell line was used as a control for the plant extract treatment. Clinically used anticancer drug, doxorubicin, was used as a control drug (for relative comparison to the plant extract) to evaluate the selective cytotoxicity of the plant extract on VNBRCA1 and HF cells. We examined the apoptosis induction by the plant extract on VNBRCA1by Annexin V/7AAD staining and flow cytometry analysis. In addition, the morphology of apoptotic nuclei of treated cells was observed by fluorescent microscopy using double fluorescent staining: Hoechst 33342 and propidium iodide (PI).
 Results: In comparison between the cytotoxicity of the plant extract and Doxorubicin on both cell lines (VBRCA1 and HF), we observed that plant extract was selectively cytotoxic against VNBRCA1 with an IC50 value of 10610 μg/mL, while Doxorubicin was discriminatorily cytotoxic against HF with an IC50 value of 0.135+/-0.09 μg/mL. We also found that the plant extract induced apoptosis VNBRCA1 in a dose-dependent manner. In addition, fluorescent microscopy revealed disintegrated nuclei of plant extract-treated cells, representing a hallmark of apoptosis.
 Conclusions: These results showed that Paramignya trimera methanolic extract selectively killed VNBRCA1 cell lines, indicating that Paramignya trimera methanolic extract may represent a potential agent for cancer treatment.

Abstract 80: Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment

Abstract Background and Aim: Are electronic cigarettes (ECs) safer than combustible tobacco? No one knows the answer yet. ECs are devices that deliver an aerosol from a heated mixture of propylene glycol, vegetable glycerin, and flavors, with or without nicotine. Short-term EC studies are mostly limited and inconclusive. Despite unknown long-term health effects, the use of ECs has increased alarmingly over the past decade. EC aerosols contain unique chemicals as well as other chemicals similar to those found in tobacco smoke. Tobacco smoking during cancer treatment has been associated with increased drug resistance and reduced overall survival rate. The awareness of the dangers of cigarette smoking and the paucity of studies evaluating the safety of ECs pose a dilemma for cancer patients who might be considering switching to ECs during chemotherapy. Here, we sought to examine the effects of EC aerosol extracts exposure on cell survival in oral cancer cells during cisplatin treatment. Methods: EC aerosol extracts were collected from two brands of ECs as previously described. Mainstream tobacco smoke (MS) extract was used as a positive control. To evaluate clonogenic survival after cisplatin treatment, oral cancer cells were exposed for 48 h to EC aerosol extracts at nicotine doses comparable to those observed in EC users. Next, cells were treated with both EC aerosol extracts and cisplatin (or vehicle-control) for another 48 h. After treatment, cells were counted and seeded in 6-well plates and colony formation was assessed two weeks later. Colonies were fixed with methanol followed by staining with 0.5% crystal violet in 25% methanol and counted manually. Only colonies with at least 50 cells were counted. Data were analyzed by Student's t-tests and one-way analysis of variance (ANOVA). Results: After cisplatin treatment, cells exposed to EC aerosol extracts showed a significant increase in clonogenic survival fraction compared to vehicle-control treated cells. Furthermore, cells exposed to EC aerosol extracts formed visibly larger colonies. An increase in clonogenic survival was also observed in MS extract-treated cells. Conclusions: Collectively, our data suggest that short-term exposure to EC aerosol can induce cisplatin resistance in oral cancer cells, thus allowing the cells to survive and form colonies. These findings are particularly important in paving the way to understanding whether ECs are a safer alternative for oral cancer patients undergoing chemotherapy. Citation Format: Jimmy Manyanga, Toral R. Mehta, Lurdes Queimado. Electronic cigarette aerosols increase clonogenic survival in oral cancer cells after cisplatin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 80.

A metabolic investigation of anticancer effect of G. glabra root extract on nasopharyngeal carcinoma cell line, C666-1.

Although a majority of nasopharyngeal carcinoma (NPC) are undifferentiated and strongly radiosensitive, many NPC patients still have troubles in recurrence. Traditional Chinese medicine (TCM) is considered as potential therapeutic drugs in NPC. However, the effect of Glycyrrhiza glabra on NPC is limited. The present study shows the decreased proliferation and high apoptosis in G. glabra root extract-treated C666-1 cells, indicating the anti-cancerous function of G. glabra in NPC. Then GC/MS-based metabolomics is employed to characterize variation of metabolomes in response to G. glabra root extract treatment. Metabolic category elaborates the higher percentage of down-regulated amino acids and lipids after G. glabra treatment. Moreover, ICA and pathway enrichment analysis further observe that glycine, serine and threonine metabolism, fatty acid biosynthesis, alanine, aspartate and glutamate metabolism, and cysteine and methionine metabolism are four important amino acid and lipid metabolisms that likely contribute to the anti-cancer effect of G. glabra in NPC. These pathways point out the seven metabolite biomarkers, glutathione, glutamine, L-alanine, glycine, L-serine, tetradecanoic acid and stearic acid. Taken together, these findings provide potential clues that anti-cancer mechanisms of G. glabra root extract are linked to the metabolic strategies and emphasize the significance of metabolic strategies against NPC.

Effects of <i>Bufei Yishen</i> formula on klotho gene expression and inflammation-related RIG-I/NF-κB pathway in cigarette smoke extract-treated BEAS-2B cells

Purpose: To investigate the anti-inflammatory effect of Bufei yishen formula on chronic obstructive pulmonary disease (COPD) and klotho expression in cigarette smoke extract (CSE)-treated BEAS-2B cells.Methods: Cigarette smoke extract (CSE) was diluted in Dulbecco’s modified Eagle’s medium (DMEM) and used to treat a COPD cell model established using BEAS-2B cells. The CSE-treated cells were transfected with klotho overexpression plasmid, and the COPD cell model was treated with Bufei yishen formula. Dexamethasone was used as positive control. Changes in the expressions of klotho, RIG-I, and NF-ĸB were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, while enzyme linked immunosorbent assay (ELISA) was used to evaluate changes in the expressions of IL-6 and TNF-α.Results: Klotho expression level was reduced in COPD model. Overexpression of klotho inhibited the expressions of IL-6, TNF-α, RIG-I, NF-κB, but bufei yishen formula increased klotho expression level and decreased the expressions of RIG-I, NF-κB, IL-6 and TNF-α. However, after CSE treatment, the anti-inflammatory effect of the formula decreased significantly when klotho expression was knocked down.Conclusion: Bufei yishen formula possesses anti-inflammatory effects which are significantly decreased after klotho knockdown. This suggests that the formula probably acts by upregulating klotho expression, thereby down-regulating the expressions of IL-6 and TNF-α via RIG-I/NF-κB pathway.Keywords: Bufei yishen formula, COPD, Klotho, Inflammation, RIG-I/NF-κB

Identification of the chemical constituents of aqueous and alcoholic Glycyrrhiza glabra root extracts and its comparative apoptotic study against human hepatocarcinoma cell line

Introdution: Hepatocellular carcinoma (HCC) is one of the most common cancers. It is the third leading cause of cancer related death. This study aimed to evaluate the phytochemical composition of ethanolic and aqueous Glycyrrhiza glabra root extracts its apoptotic effects against human hepatocarcinoma HepG2 cell line. Methods: In the current experimental study, the phyochemical composition of aqueous and ethanolic Glycyrrhiza glabra extract was evaluated by GC/MS method. The HepG2 cells were grown in RPMI1640 supplemented with 10 % (v/v) FBS at 37°C with 5 %carbon dioxide (CO2) atmosphere. The cytotoxicity activities of extracts were measured using MTT after 24 hours. To evaluate the ability of extracts to trigger apoptosis in HepG2 cells, the Hosest8778 staining method was carried out. Results: The phytochemical data of aqueous extract revealed 18 compounds, including Dodecane (13.34%) and 2',6'-Dihydroxyacetophenone bis (trimethylsilyl) ether (12.48%) as the major constitutes. The chemical composition of ethanolic extract showed 17 compounds, including Pentadecane (7.78%), 2,6,11,15-Tetramethylhexadecane (4.67%) as the prominent compounds. The MTT results revealed that the extract-treated cells had a dose-related toxicity effects. The results of Hoechst 33258 staining showed that the nuclear DNA fragmentation was increased in the ethanolic extract treatment compared to aqueous extract. Conclusion: The ethanolic extract of Glycyrrhiza glabra possesses more apoptotic effect on cancer cells and further researches are needed for investigating its potential mechanism.

<i>Artemisia anomala</i> extracts enhance the viability and antioxidation capacity of human keratinocytes

Purpose : To investigate the effect of extracts of Artemisia anomala S. Moore tissues on viability, apoptosis and antioxidant capacity of human keratinocytes. Methods : Human keratinocyte cell line HaCaT were treated with extracts of A. anomala for 12 h or 24 h. Cell viability, level of reactive oxygen species (ROS), and incidence of apoptosis were measured by flow cytometry. Levels of mRNA and key proteins in the mitogen-activated protein kinase (MAPK) pathway were determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Key proteins of caspase pathways were assessed by western blot. The influence of the extract on the MAPK pathway was further probed by treating cells with MAPK activator in the presence and absence of the extract. Results : Treatment of cells with extracts of A. anomala enhanced viability and reduced apoptosis in a time-dependent manner, and increased ROS level, compared with control. mRNA and protein expressions of c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK), and p38 MAPK decreased in extract-treated cells. The extracts also reversed the inhibitory effects of the MAPK pathway activator, actinomycin, on cell viability and ROS, and inhibited protein-cleaved caspase-8 and cleaved caspase-3. Conclusion : A. anomala extract increases cell viability and antioxidant capacity via inactivation of MAPK pathway, and also inhibits cell apoptosis via inactivation of caspase pathways. Hence, the extract may serve as a promising drug for the treatment of psoriasis. Keywords : Artemisia anomala , MAPK pathway, Anti-oxidation, Keratinocyte, Psoriasis

Antioxidant Potential and Antitumour Activities of Nendran Banana Peels in Breast Cancer Cell Line

The present study was aimed to evaluate the antioxidant and antitumor activities of the aqueous methanol extract of Nendran banana peel as a valuable source for bioactive compounds against cancer. Free radical scavenging activity of the peel extracts was evaluated using 1,1-diphenyl-2-picrylhydrazyl and ferric reducing antioxidant power assay. The antitumor activity on MCF-7 breast cancer cell line was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and ethidium bromide/acridine orange staining assay. Extract of banana peel was more efficient in reducing ferric ion with the increase in concentration and exhibited IC50 180.33 µg/ml, free radical inhibition activity under 2,2-diphenyl-1-picrylhydrazyl hydrate assay. Flavonoid content was high (51.47 µg/ml) in the peel extract, which could have contributed to the antioxidant activity. The antioxidant enzyme concentration such as ascorbate peroxidase, catalase, peroxidase and superoxide dismutase were found higher in the fruit pulp than peel. As the concentration of extract increased from 20 to 200 μg/ml, the cell viability of MCF-7 decreased from 91.14 to 24.7%. The IC50 values of plant extract-treated cells (120.6 μg/ml) showed typical apoptotic and necrotic morphological features such as condensed nuclei, membrane blebbing and formation of apoptotic bodies. This indicated the efficiency of sample against breast cancer with the highest inhibition of growth.