I3M inhibits oral cancer cell proliferation and induces apoptosis.

<p>(A) The inhibitory effects of indirubin and I3M on Cal-27 and HSC-3 cell proliferation. Cells were treated for 24 or 48 h with various concentrations of indirubin or I3M. Cell proliferation was analyzed by MTT assay (top) and cell counting using a hemocytometer (bottom). The data are presented as the mean ± S. D. values; asterisks denote a statistically significant difference (<i>P</i><0.05). (B) Cal-27 cells (10⁵) were treated with 10 µM I3M for 24 h, and the percentage of apoptotic cells was determined using the Annexin V-FITC Apoptosis Detection kit. The data are presented as the mean ± S.D. values (n = 3); asterisks denote a statistically significant difference (<i>P</i><0.05) between the I3M treatment and control groups. (C) Immunoblots of protein extracts (30 µg) isolated from the cytosolic and mitochondria fractions of Cal-27 cells treated with 0, 2.5, 5, or 10 µM I3M for 24 h. The results shown are representative of six independent experiments.</p

I3M suppresses 4-NQO/arecoline-induced oral cancer in mice.

<p>(A) In the mice experiments, the body weight were not shown the significant different among the four groups until 8 th week. After the I3M experiments, the body weight of the carrier group was significantly lower than any other groups at 28 th week (<i>p</i> = 0.031, n = 10). (B) Oral cancer lesions were induced on the tongues of the mice with 0.5 mg/mL arecoline and 0.2 mg/mL 4-NQO. The table shows tumor numbers and sizes from four groups (10 mice/group/sampling).</p

I3M induces cell cycle arrest.

<p>(A) Cal-27 cells were treated with I3M (0, 2.5, 5, or 10 µM) for 24 h. Following treatment, the cells were collected, fixed with methanol, stained with propidium iodide, and analyzed using flowcytometry. The data for each sample represent the percentage of cells found in G0/G1, S, and G2/M phases of the cell cycle. (C) Immunoblots showing the expression of cell cycle-related proteins in I3M-treated Cal-27 cells. Total cell lysates were prepared after 24 h treatment with I3M (0, 2.5, 5, or 10 µM). The expression of p53 and p21 was determined using immunoblotting. <i>β</i>-actin was used as the loading control in this study. Experiments were repeated three times with similar results.</p

I3M inhibits cancer cell migration and invasion.

<p>(A) Incubation of cells in the absence or presence of 10 µM indirubin or I3M was carried out for 0, 24, or 48 h. The cells were photographed under phase-contrast microscopy (100× magnification). The data are shown as the percentage of inhibition; asterisks indicate a statistically significant difference (<i>P</i><0.05) between treatment and control groups. (B) Cells (10⁴) were plated in the upper chamber with I3M (0, 2.5, 5, or 10 µM) and allowed to undergo migration for 24 or 48 h. Quantification of cells in the lower chamber was carried out by counting cells under ×200 magnification. The percentage of inhibition and column mean values were derived from 3 independent experiments. The asterisks indicate a statistically significant difference (<i>P</i><0.05) between treatment and control groups. (C) Immunoblots showing changes in the levels pp38, FAK, MMP-9 and uPA proteins associated with migration and invasion in Cal-27 cells following 10-µM I3M treatment (n = 3).</p

Identification of survivin is a target of I3M treatment and associate with Caspase-3/7 and -9 activity.

<p>(A) A demonstration of time-dependent survivin downregulation following 10-µM I3M treatment for various durations. Data are presented as the mean ± S. D. values (n = 3); asterisks indicate a statistically significant difference (<i>P</i><0.05) between the treatment and control groups. (B) The dose-dependent of downregulation of survivin following I3M treatment for 12 h. The data are presented as the mean ± S. D. values (n = 3); asterisks indicate a statistically significant difference (<i>P</i><0.05) between treatment and control groups. (C) Cal-27 cells were incubated with I3M (10 µM) for 0, 6, 12, 24, or 48 h. Shown are the relative gene expression levels for each sample in the treatment group (n = 6). The bold lines denote the mean related percentage of the individual treatment groups. T/C: Treated group (6, 12, 24, or 48 h)/control group (0 h). (D) A demonstration of time-dependent Caspase-3/7 and -9 upregulations following 10-µM I3M treatment for 12, 24, and 48 h.</p