Synthesis and biological evaluation of natural products and their analogs as new cancer chemotherapeutic agents

Ph.DDOCTOR OF PHILOSOPH

Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

10.1371/journal.pone.0076754PLoS ONE810-POLN

Generation of Reactive Oxygen Species by Polyenylpyrroles Derivatives Causes DNA Damage Leading to G2/M Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma Cells

10.1371/journal.pone.0067603PLoS ONE86-POLN

The proposed mechanism for compound 1a-mediated apoptosis in human oral squamous cell carcinoma cells.

<p>The proposed mechanism for compound 1a-mediated apoptosis in human oral squamous cell carcinoma cells.</p

Compound 1a-induced apoptosis and DNA damage through cytochrome P450.

<p>(<b>A</b>) OEC-M1 cells were incubated with NAC (10 mM), ketoconazole (KTZ, 50 µµM), diisothiocyanatostilbene-disulfonic acid (DIDS, 10 µM), apocynin (0.3 µM), rotenone (1 µM), L-NAME (500 µM), allopurinol (100 µM), indomethacin (50 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle (0.1% DMSO) treatment for 24 h. The cell cycle distribution was determined by flow cytometry after PI-staining of nuclei. (<b>B</b>) OEC-M1 cells were incubated with KTZ (50 µM) and NAC (10 mM) in the presence of 2′,7′-dichlorofluorescein diacetate (2 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle (0.1% DMSO) treatment for 30 min, and then the ROS production was measured. (<b>C</b>) OEC-M1 cells were incubated with NAC (10 mM) or ketoconazole (KTZ, 50 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle treatment for the time as indicated. AP sites of DNA lesions were analyzed by a DNA damage quantification kit. (<b>D</b>) OEC-M1 cells were treated with NAC (10 mM) or ketoconazole (KTZ, 50 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle (0.1% DMSO) treatment for 2 or 16 h. The expression levels of p-chk2 (2 h), cyclin B1, p21, and p-cdc2 were measured by western blotting. (<b>E</b>) OEC-M1 cells were incubated with resveratrol (100 µM) or itraconazole (10 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle treatment for 24 h. The cell cycle distribution was determined by flow cytometry after PI-staining of nuclei. (<b>F</b>) Untreated OEC-M1 cells, cytochrome P450 1A1 siRNA transfected OEC-M1 cells, and control siRNA transfected OEC-M1 cells were incubated with compound <b>1a</b> (5 µM) or vehicle for 24 h and then the cytochrome P450 1A1 expression were measured by western blotting. (<b>G</b>) Untreated OEC-M1 cells, cytochrome P450 1A1 siRNA transfected OEC-M1 cells, and control siRNA transfected OEC-M1 cells were incubated with compound <b>1a</b> (5 µM) or vehicle for 30 min and then the ROS production was measured. (<b>H</b>) Untreated OEC-M1 cells, cytochrome P450 1A1 siRNA transfected OEC-M1 cells, and control siRNA transfected OEC-M1 cells were incubated with compound <b>1a</b> (5 µM) or vehicle for 24 h. The cell number was determined by trypan blue staining. In (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>E</b>), (<b>G</b>) and (<b>H</b>), the data are expressed as mean ± SD of three independent experiments, while, in (<b>D</b>) and (<b>F</b>), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *indicates a significant difference at the level of <i>p</i><0.05.</p

Characterization of compound 1a-induced apoptotic cell death.

<p>(<b>A</b>) Cells were seeded in 96-well plates, treated with compound <b>1a</b> (1.25 ∼ 40 µM) or vehicle (0.1% DMSO) for 24 and 48 h. Cell viability was measured by A<i>lamarBlue</i>® assays. The data were expressed as mean ± SD; n = 3. (<b>B</b>) OEC-M1 and HSC-3 cells were treated with compound <b>1a</b> (5 µM) or vehicle for 24 h, and the DNA breaks were analyzed by flow cytometry based TUNEL assay. The results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *indicates a significant difference at the level of <i>p</i><0.05. (<b>C</b>) OEC-M1 cells were treated with various concentrations of compound <b>1a</b> as indicated, Z-VAD-FMK (50 µM) plus compound <b>1a</b> (5 µM) or vehicle for 24 h. The cell cycle distribution was determined by flow cytometry after PI-staining of the nuclei. The data were expressed as mean ± SD; n = 3. *indicates a significant difference at the level of <i>p</i><0.05. (<b>D</b>) OEC-M1 cells were treated with compound <b>1a</b> (5 µM) or vehicle for the time as indicated. The expression of p-chk2, p53 and p21 were measured by western blotting. The results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. * and **indicate a significant difference at the level of <i>p</i><0.05 and <i>p</i><0.01 respectively.</p

Anticancer Activity of Gymnoconjugatin Derivatives against Human Oral Cancer Cells.

a<p>The concentration of the tested compound is 20 µM; the concentration of paclitaxel is 50 nM.</p>b<p>The cell viability are expressed in % of control as mean ± SD of three independent experiments.</p

Compound 1a-induced apoptosis is dependent on oxidative stress.

<p>(<b>A</b>) OEC-M1 cells were incubated with 2′,7′-dichlorofluorescein diacetate (2 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle (0.1% DMSO) treatment for the time indicated. The relative mean fluorescence intensity (MFI) of 2′,7′-dichlorofluorescein was detected at an excitation wavelength of 485 nm and an emission wavelength of 530 nm with a fluorometer; n = 3. (<b>B</b>) OEC-M1 cells were incubated with <i>N</i>-acetyl-cysteine (NAC) (10 mM) or Z-VAD-FMK (50 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle treatment for 24 h. The cell cycle distribution was determined by flow cytometry after PI-staining of nuclei. The data were expressed as mean ± SD; n = 3. (<b>C</b>) OEC-M1 cells were incubated with NAC (10 mM), Z-VAD-FMK (50 µM), or DPI (25 µM) for 30 min, followed by compound <b>1a</b> (5 µM) or vehicle treatment for 16 h. The mitochondrial membrane potential was analyzed by flow cytometry using DiOC₂(3) staining. The data are expressed as mean ± SD of MFI; n = 3. *indicates a significant difference at the level of <i>p</i><0.05.</p

Inhibition of human oral cancer xenografts growth in vivo by compound 1a.

<p>(<b>A</b>) OEC-M1 tumor-bearing mice were administered s.c. with vehicle control (◊), 15 mg/kg (△), or 30 mg/kg (×) compound <b>1a</b> on days 0–4 for 5 days. The figure showed the relative tumor volume of control and compound <b>1a</b>-treated groups. (<b>B</b>) Caspase 3 activity of tissues from OEC-M1 tumor-bearing mice (on day 28 after inoculation). (<b>C</b>) The changes of body weight of the tumor-bearing mice. Body weight was measured for each animal every three days starting from the day of the initial compound <b>1a</b> treatment. All results are given as means ± SD; n = 6 for each group.*indicates a significant difference at the level of <i>p</i><0.05.</p

Compound 1a induces mitochondrial death pathway and caspase-dependent apoptosis.

<p>(<b>A</b>) Cells were stimulated with compound <b>1a</b> (5 µM) or vehicle (0.1% DMSO) for the time indicated, followed by DiOC₂(3) staining. The mitochondrial membrane potential was analyzed by flow cytometry. The results were expressed as mean ± SD of mean fluorescence intensity (MFI); n = 3. (<b>B</b>) OEC-M1 cells were stimulated with compound <b>1a</b> (5 µM) or vehicle for 16 h, the cytochrome <i>c</i> in mitochondria was measured by flow cytometry. The results were expressed as mean ± SD of MFI; n = 3. (<b>C</b>) The cytochrome <i>c</i> in mitochondrial fraction and cytosolic fraction were measured by western blotting. Voltage-dependent anion channel (VDAC) and actin were used as a marker protein for mitochondria and cytosol respectively. One of three repeated experiments was shown. (<b>D</b>) OEC-M1 cells were treated with various concentration of compound <b>1a</b> as indicated or vehicle for 24 h. The expression levels of Bcl-2, Bax and Bak were measured by western blotting. (<b>E</b>) OEC-M1 cells were treated with various concentration of compound <b>1a</b> as indicated or vehicle for 24 h. The expression levels of caspase 3 precursor, cleaved caspase 3, caspase 9 precursor, caspase 8 precursor, PARP and β-catenin were measured by western blotting. In (<b>D</b>) and (<b>E</b>), the results are representative of those obtained in three different experiments and the histogram shows the quantification expressed as the mean ± SD. *indicates a significant difference at the level of <i>p</i><0.05 compared to control group.</p