HCT-116RC cells treated with either control media, 0.5 mM DOC (deoxycholate), control siRNA, or maspin-specific siRNA for 24 hours and the percentage of apoptosis ± SEM determined for each experimental group. The single asterisk indicates statistically significant differences compared to untreated cells in control media. Treatment of cells with 0.5 mM DOC served as a control to ensure that the cells had maintained their apoptosis resistance to the same apoptosis-inducing agent that was used to develop the resistant cells over ~40 weeks of persistent exposure. There was no significant increase in the % apoptosis after treatment of cells with 0.5 mM DOC.
Notes: *Control siRNA treatment resulted in significantly more apoptosis than control cells and DOC-treated cells; **Maspin siRNA treatment resulted in significantly more apoptosis than both control cells and DOC-treated cells; ^#% apoptosis induced by the specific maspin siRNA probe was significantly higher than that produced by the siRNA control.
Abbreviations: siRNA, small interfering RNA; SEM, standard error of the mean; DOC, deoxycholate.

(A) Western blots of maspin expression from HCT-116RC cells treated with 0.5 mM DOC, siRNA control probe, or siRNA maspin probe for 24 hours. (B) Relative protein expression levels were obtained using densitometry. DOC treatment was used as an internal control to ensure that the resistant cells still maintained the same high level of maspin expression and could not be further induced by DOC. Routine staining of the entire gel with Brilliant blue indicated that all lanes received the same amount of protein.
Note: *Reduction in maspin protein levels after incubation with the specific siRNA maspin probe was statistically significant compared with untreated control cells (P = 0.0078).
Abbreviations: siRNA, small interfering RNA; SEM, standard error of the mean; DOC, deoxycholate.

Composite of images of colonic tissue from a patient with ulcerative colitis and adenocarcinoma stained for maspin expression. (A) Non-neoplastic mucosa taken adjacent to the cancer on the proximal side. (B) Non-neoplastic mucosa taken adjacent to the cancer on the distal side. (C) Non-neoplastic mucosa taken 3 cm away from the cancer on the proximal side. (D) Non-neoplastic mucosa taken 9 cm away from the cancer on the distal side. (E) Higher magnification of representative non-neoplastic mucosa taken 3 cm away from the cancer on the distal side; note the predominately cytoplasmic staining of maspin. (F) Note the increased overall expression of maspin in the adenocarcinoma; prominent nuclear staining of maspin is evident.
Notes: Images A–D were taken with a 4× objective lens; images E and F were taken with a 20× objective lens; all images counterstained with hematoxylin.

Variability in maspin expression in non-neoplastic colonic mucosa obtained from normal subjects and patients with colonic tumors (4× objective). (A) Normal mucosa obtained from a healthy patient during routine colonoscopy. This image shows a low overall expression of maspin; (B) normal mucosa obtained from a healthy patient during routine colonoscopy. This image shows focal staining in individual cells (arrows); (C) normal mucosa obtained from a colon resection from a patient who had a sarcoma surgically removed. The sarcoma was external to the body wall and the patient had received no prior radiation or chemotherapy. This image shows low overall staining predominantly at the top of the crypts (arrows); (D) region of colon 2 cm proximal to a large tubulovillous adenoma. This image shows a low overall expression of maspin; (E) region of colon 2 cm proximal to a large tubulovillous adenoma. This image shows a low overall expression of maspin; (F) region of colon near the distal margin of a colon resection from a patient with a colon cancer. This image shows low overall staining predominantly at the top of the crypts (arrows).

Histogram displaying the average maspin score ± SEM in the non-neoplastic mucosa at different distances from the colonic neoplasms of 10 patients with a large adenoma (A) or cancer (CA). Samples of the non-neoplastic mucosa were taken adjacent to the tumor on the proximal and distal sides (P0, D0), 2 cm away on the proximal and distal side (P2, D2) and near the surgical margins (PMAX, DMAX).
Abbreviation: SEM, standard error of the mean.

Increased maspin expression in polyps (brown color) compared with minimal expression in adjacent non-neoplastic colonic mucosa (see arrows). (A) Adenomatous polyp; (B) adenomatous polyp; (C) hyperplastic polyp; (D) adenomatous polyp (4× objective).

Molecular signaling pathways that may be activated after persistent exposure of cells to hydrophobic bile acids. Since hydrophobic bile acids can perturb cellular membranes, surface molecules can be activated that generate reactive oxygen and nitrogen species. This can cause oxidative DNA damage and the activation of genes associated with the stress response and the activation of transcription factors that turn on genes associated with cell survival. These survival genes include maspin, classic anti-apoptotic genes that are NF-κB-activated, and autophagy-related genes, such as beclin. In order for cells with DNA damages to proliferate, they must evade cell death pathways. Maspin may inhibit these pro-apoptotic pathways through interaction with proteins (eg, GST, HSP70) known to interact with maspin and to inhibit apoptosis. Future studies, designated by question marks, indicate possible interactions with apoptosis regulatory proteins, caspases (executioners of apoptosis) and proteins associated with the pro-survival NF-κB and autophagic pathways. Some of these potential maspin-target protein interactions may result in activation or inhibition of the target proteins.
Abbreviations: ATF-2, activating transcription factor 2; GST, glutathione S-transferase; HSP70, heat shock protein 70; NF-κB, redox-sensitive transcription factor; NFKB1, nuclear factor NF-κB p105 subunit; NKX3-1, product of the homeobox gene that functions as a transcription factor; p53, tumor protein 53; RNS, reactive nitrogen species; ROS, reactive oxygen species; SP1, transcription factor that responds to physiological and pathological stimuli.