Investigation of cell cytotoxic activity and molecular mechanism of 5β,19-epoxycucurbita-6,23(E)-diene-3β,19(R),25-triol isolated from Momordica charantia on hepatoma cells
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Context Momordica charantia L. (Cucurbitaceae), known as bitter melon, is an edible fruit cultivated in the tropics. In this study, an active compound, 5β,19-epoxycucurbita-6,23(E)-diene-3β,19(R),25-triol (ECDT), isolated from M. charantia was investigated in regard to its cytotoxic effect on human hepatocellular carcinoma (HCC) cells.Objective To examine the mechanisms of ECDT-induced apoptosis in HCC cells.Materials and methods The inhibitive activity of ECDT on HA22T HCC cells was examined by MTT assay, colony formation assay, wound healing assay, TUNEL/DAPI staining, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and JC-1 dye. HA22T cells were treated with ECDT (5, 10, 15, 20 and 25 μM) for 24 h, and the molecular mechanism of cells apoptosis was examined by Western blot. Cells treated with vehicle DMSO were used as the negative control.Results ECDT inhibited the cell proliferation of HA22T cells in a dose-dependent manner. Flow cytometry showed that ECDT treatment at 10–20 μM increased early apoptosis by 10–14% and late apoptosis by 2–5%. Western blot revealed that ECDT treatment activated the mitochondrial-dependent apoptotic pathway, and ECDT-induced apoptosis was mediated by the caspase signalling pathway and activation of JNK and p38MAPK. Pre-treatment of cells with MAPK inhibitors (SB203580 or SP600125) reversed the ECDT-induced cell death, which further supported the involvement of the p38MAPK and JNK pathways.Discussion and conclusions Our results indicated that ECDT can induce apoptosis through the p38MAPK and JNK pathways in HA22T cells. The findings suggested that ECDT has a valuable anticancer property with the potential to be developed as a new chemotherapeutic agent for the treatment of HCC.Keywords:
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Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry Linda Rodgers This protocol was adapted from “Flow Cytometry,” Chapter 16, in Cells (eds. Spector et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 1998. This threevolume set is now out of print; however, some of the microscopy methods were republished in Basic Methods in Microscopy, by David L. Spector and Robert D. Goldman. INTRODUCTION This protocol describes a method for quantitative measurement of DNA in tissue culture cells using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. After staining, cells can be stored frozen for later sorting or analysis. MATERIALS Reagents Boiled RNase A, 10 mg/ml Citrate buffer DMSO (Optional, see Step 5) DNA staining/lysis solution (For PI staining; see Step 2.) NST-DAPI buffer (For DAPI staining; see Step 2.) Equipment Flow cytometer Nylon mesh, 35-μm pore size (Small Parts, Inc.) Page 1 of 3 Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry
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The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane. Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane. However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells. This protocol describes Annexin V binding and PI uptake followed by flow cytometry to detect and quantify apoptotic and necrotic cells.
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None. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Nuclear staining with haematoxylin, DAPI and propidium iodide (PI) in archival formalin- and methacarn-fixed placental tissues. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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The interaction between pathogenic microbes and host cells is crucial for studies on infections and tumor cell biology . The kinetic studies of cell cycle are of significance for understanding the mechanisms mediated by microbes versus host cells .In this paper ,a novel method for cell cycle analysis in mouse splenic cells by Ki67 and 4′,6‐diamidino‐2‐phenylindole (DAPI) or propidium iodide (PI) co‐staining is introduced .
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Objective: To investigate the profile of 5-FU-induced apoptosis in tumor cell line and evaluate the usefulness of Annexin V and propidium iodide double staining for detecting apoptosis. Methods: Colon cancer cell line colo320 was incubated with 5-FU of different concentration for 2, 4,6, 8 and 12h respectively, apoptosis was measured using Hoechst33342 staining and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. Results: Apoptosis could be induced in colo 320 cell line by 5-FU,the percentage of AnnexinⅤ~+PI~-cells increased after being treated with 80ug/ml 5-FU for 4 h,both apoptotic and necrotic cell increased at 12h.Apoptotic cells were also identified by Hoechst33342 staining. Conclusion: Apoptosis induced by 5-Fu is time and dose dependent.Annexin V and PI double staining is a specific, sensitive, and quantitative method for analyzing apoptotic cells.
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This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium Iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.
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PURPOSE Leukemia causes tremendous human mortality especially in children and young adults. This study was undertaken to investigate the anticancer effects of Solanine against the normal human NCI-H526 and human leukemia AML-193 cell lines. METHODS Cell proliferation was determined by MTT assay. DAPI and annexin V/propidium iodide (PI) assays were used for the determination of apoptosis. The expression analysis was done by qRT-PCR. Protein concentrations were determined by western blot analysis. RESULTS DAPI staining showed that Solanine causes nuclear morphological changes. The annexin V/PI staining showed that Solanine increased the leukemia apoptotic cell death dose-dependently. The expression of Bax was increased while of Bcl-2 was decreased. The qRT-PCR analysis showed that microRNA (miR)-16 was significantly (p<0.05) downregulated in leukemia AML-193 cells as compared to normal NCI-H526 cells. CONCLUSION Taken together, these results showed that Solanine inhibits the proliferation of leukemia cells via induction of apoptosis and modulation of miR-16/Bcl-2 axis.
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