6 research outputs found
Extra- and intracellular formation of reactive oxygen species (ROS) and of lipid peroxides in HT-29 cells after treatment with IAE.
<p>A, Extracellular formation of ROS in full cell culture medium was kinetically detected using the ROS-sensitive CellROX Orange fluorogenic probe. Data are expressed as mean ± SEM (n = 8). B, Intracellular ROS was detected by flow cytometry of HT-29 cells stained with CellROX Orange after treatment for 24 h. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). C, Intracellular lipid peroxidation was detected by flow cytometry of cells treated for 24 h using the Click-iT technology. Increasing fluorescence intensities are a result of enhanced lipid peroxidation upon treatment. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). D, Intracellular lipid peroxidation was visualized by fluorescence microscopy (green, lipid peroxides; blue, nucleus). Scale bars, 25 μm. n.s. not significant, **p≤0.01, ***p≤0.001 vs. control.</p
Cell cycle analysis of HT-29 colon carcinoma cells after treatment with 30 μg/ml IAE for 24 h.
<p>A, Cell cycle was analyzed by flow cytometry of propidium iodide stained cells. Histograms (top) show one representative experiment for each treatment condition. Bar plots (bottom) show percent of cell population in apoptotic SubG1, G0/G1, S and G2/M phases of the cell cycle and are expressed as mean ± SEM (n = 3). *p≤0.05, ***p≤0.001 vs. control. B, Whole cell lysates were analyzed for the expression of cyclin A2, cyclin D3, CDK2, CDK4, CDK6 and GAPDH proteins by immunoblotting using specific antibodies.</p
Activation of apoptosis signaling pathway in colon carcinoma cells after treatment with IAE.
<p>A, HT-29 and T84 cells were treated for 24 h. Enzymatic activation of caspases 2, 3/7, 6, 8 and 9 was determined by use of luminescence-based assays. Data are normalized to control treatment and are expressed as mean ± SEM (n = 4). B, Whole cell lysates from HT-29 cells treated for 24 h were analyzed for the expression of total and cleaved proteins of caspase 3, caspase 9 and PARP by immunoblotting. Numbers indicate densitometric ratios of the cleaved to total proteins normalized to control treatments. C, Fluorescence microscopy of HT-29 cells treated for 24 h. Cleaved caspase 3 was labeled green, F-actin red and the nucleus blue. Scale bars, 25 μm. D, HT-29 cells were treated for 6 h. DNA fragmentation was detected through accumulation of cytoplasmic BrdU-labeled DNA by ELISA. Data are normalized to control treatment and are expressed as mean ± SEM (n = 5). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control.</p
Cytotoxicity test and evaluation of inhibition of tumor growth by treating HT-29 tumor xenograft mouse models with IAE.
<p>Athymic nude mice were ectopically implanted with 5 million HT-29 cells in the flank and orally gavaged bidaily by 50 mg/kg IAE or vehicle for 4 weeks. A, Cytotoxicity was tested for colon cancer and primary colon cells (IC<sub>50</sub> = 25.82 μg/mL (HT-29); IC<sub>50</sub> = 36.12 μg/mL (CCD 841 CoN)). Data are expressed as mean ± SD (n = 4). B, Body weight during entire experiment. C, Tumor volume during entire experiment and images of exemplary xenografts of untreated and IAE-treated mice at end point. D, Tumor weight at the end of the study. Data are expressed as mean ± SEM (n = 18). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control (one-tailed t test).</p
Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.
<p>Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.</p
Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria
A known (<b>1</b>) and a structurally related new natural
product (<b>2</b>), both belonging to the amorfrutin benzoic
acid class, were isolated from the roots of <i>Glycyrrhiza foetida</i>. Compound <b>1</b> (amorfrutin B) is an efficient agonist
of the nuclear peroxisome proliferator activated receptor (PPAR) gamma
and of other PPAR subtypes. Compound <b>2</b> (amorfrutin C)
showed comparably lower PPAR activation potential. Amorfrutin C exhibited
striking antiproliferative effects for human colorectal cancer cells
(HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7)
cells (IC<sub>50</sub> values ranging from 8 to 16 μM in these
cancer cell lines). Notably, amorfrutin C (<b>2</b>) showed
less potent antiproliferative effects in primary colon cells. For
HT-29 cells, compound <b>2</b> induced G0/G1 cell cycle arrest
and modulated protein expression of key cell cycle modulators. Amorfrutin
C further induced apoptotic events in HT-29 cells, including caspase
activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization,
and formation of reactive oxygen species. Mechanistic studies revealed
that <b>2</b> disrupts the mitochondrial integrity by depolarization
of the mitochondrial membrane (IC<sub>50</sub> 0.6 μM) and permanent
opening of the mitochondrial permeability transition pore, leading
to increased mitochondrial oxygen consumption and extracellular acidification.
Structure–activity-relationship experiments revealed the carboxylic
acid and the hydroxy group residues of <b>2</b> as fundamental
structural requirements for inducing these apoptotic effects. Synergy
analyses demonstrated stimulation of the death receptor signaling
pathway. Taken together, amorfrutin C (<b>2</b>) represents
a promising lead for the development of anticancer drugs