Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

PURPOSE: The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS: Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS: A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/ß-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS: High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic ß-Catenin.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Dysplastic Aberrant Crypt Foci: Biomarkers of Early Colorectal Neoplasia and Response to Preventive Intervention.

The discovery of aberrant crypt foci (ACF) more than three decades ago not only enhanced our understanding of how colorectal tumors form, but provided new opportunities to detect lesions prior to adenoma development and intervene in the colorectal carcinogenesis process even earlier. Because not all ACF progress to neoplasia, it is important to stratify these lesions based on the presence of dysplasia and establish early detection methods and interventions that specifically target dysplastic ACF (microadenomas). Significant progress has been made in characterizing the morphology and genetics of dysplastic ACF in both preclinical models and humans. Image-based methods have been established and new techniques that utilize bioactivatable probes and capture histologic abnormalities in vivo are emerging for lesion detection. Successful identification of agents that target dysplastic ACF holds great promise for intervening even earlier in the carcinogenesis process to maximize tumor inhibition. Future preclinical and clinical prevention studies should give significant attention to assessing the utility of dysplastic ACF as the earliest identifiable biomarker of colorectal neoplasia and response to therapy.See all articles in this Special Collection Honoring Paul F. Engstrom, MD, Champion of Cancer Prevention.

Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas.

OBJECTIVE: The response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those with colorectal tumours. Sulindac and/or atorvastatin was administered to Apc+/Min-FCCC mice with known tumour-bearing status at treatment initiation. DESIGN: Male mice (6-8 weeks old) underwent colonoscopy and received control chow or chow with sulindac (300 ppm), atorvastatin (100 ppm) or sulindac/atorvastatin. Tissues were collected from mice treated for 14 weeks (histopathology) or 7 days (gene expression). Cell cycle analyses were performed on SW480 colon carcinoma cells treated with sulindac, atorvastatin or both. RESULTS: The multiplicity of colorectal adenomas in untreated mice bearing tumours at baseline was 3.6-fold higher than that of mice that were tumour free at baseline (P=0.002). Atorvastatin completely inhibited the formation of microadenomas in mice that were tumour free at baseline (P=0.018) and altered the expression of genes associated with stem/progenitor cells. Treatment of tumour-bearing mice with sulindac/atorvastatin led to a 43% reduction in the multiplicity of colorectal adenomas versus untreated tumour-bearing mice (P=0.049). Sulindac/atorvastatin increased the expression of Hoxb13 and Rprm significantly, suggesting the importance of cell cycle regulation in tumour inhibition. Treatment of SW480 cells with sulindac/atorvastatin led to cell cycle arrest (G0/G1). CONCLUSIONS: The tumour status of animals at treatment initiation dictates response to therapeutic intervention. Atorvastatin eliminated microadenomas in tumour-free mice. The tumour inhibition observed with Sul/Atorva in tumour-bearing mice was greater than that achieved with each agent.

Imaging Matrix Metalloproteases in Spontaneous Colon Tumors: Validation by Correlation with Histopathology.

The use of fluorescent probes in conjunction with white-light colonoscopy is a promising strategy for improving the detection of precancerous colorectal lesions, in particular flat (sessile) lesions that do not protrude into the lumen of the colon. We describe a method for determining the sensitivity and specificity of an enzymatically activated near-infrared probe (MMPSense680) for the detection of colon lesions in a mouse model (APC+/Min-FCCC) of spontaneous colorectal cancer. Fluorescence intensity correlates directly with the activity of matrix metalloproteinases (MMPs). Overexpression of MMPs is an early event in the development of colorectal lesions. Although the probe employed serves as a reporter of the activity of MMPs, our method can be applied to any fluorescent probe that targets an early molecular event in the development of colorectal tumors.

Plecanatide-mediated activation of guanylate cyclase-C suppresses inflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice.

AIM: To evaluate the effect of orally administered plecanatide on colorectal dysplasia in Apc+/Min-FCCC mice with dextran sodium sulfate (DSS)-induced inflammation. METHODS: Inflammation driven colorectal carcinogenesis was induced in Apc+/Min-FCCC mice by administering DSS in their drinking water. Mice were fed a diet supplemented with plecanatide (0-20 ppm) and its effect on the multiplicity of histopathologically confirmed polypoid, flat and indeterminate dysplasia was evaluated. Plecanatide-mediated activation of guanylate cyclase-C (GC-C) signaling was assessed in colon tissues by measuring cyclic guanosine monophosphate (cGMP) by ELISA, protein kinase G-II and vasodilator stimulated phosphoprotein by immunoblotting. Ki-67, c-myc and cyclin D1 were used as markers of proliferation. Cellular levels and localization of ß-catenin in colon tissues were assessed by immunoblotting and immunohistochemistry, respectively. Uroguanylin (UG) and GC-C transcript levels were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). A mouse cytokine array panel was used to detect cytokines in the supernatant of colon explant cultures. RESULTS: Oral treatment of Apc+/MinFCCC mice with plecanatide produced a statistically significant reduction in the formation of inflammation-driven polypoid, flat and indeterminate dysplasias. This anti-carcinogenic activity of plecanatide was accompanied by activation of cGMP/GC-C signaling mediated inhibition of Wnt/ß-catenin signaling and reduced proliferation. Plecanatide also decreased secretion of pro-inflammatory cytokines (IL-6, IL1 TNF), chemokines (MIP-1, IP-10) and growth factors (GCSF and GMCSF) from colon explants derived from mice with acute DSS-induced inflammation. The effect of plecanatide-mediated inhibition of inflammation/dysplasia on endogenous expression of UG and GC-C transcripts was measured in intestinal tissues. Although GC-C expression was not altered appreciably, a statistically significant increase in the level of UG transcripts was detected in the proximal small intestine and colon, potentially due to a reduction in intestinal inflammation and/or neoplasia. Taken together, these results suggest that reductions in endogenous UG, accompanied by dysregulation in GC-C signaling, may be an early event in inflammation-promoted colorectal neoplasia; an event that can potentially be ameliorated by prophylactic intervention with plecanatide. CONCLUSION: This study provides the first evidence that orally administered plecanatide reduces the multiplicity of inflammation-driven colonic dysplasia in mice, demonstrating the utility for developing GC-C agonists as chemopreventive agents.

Differential response of flat and polypoid colitis-associated colorectal neoplasias to chemopreventive agents and heterocyclic amines.

Individuals with ulcerative colitis face an increased risk of developing colorectal cancer and would benefit from early chemopreventive intervention. Results from preclinical studies in the mouse model of dextran sulfate sodium-induced colitis demonstrate that flat and polypoid colitis-associated dysplasias arise via distinct genetic pathways, impacted by the allelic status of p53. Furthermore, flat and polypoid dysplasias vary in their response to induction by the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and inhibition by 5-aminosalicylic acid, a common therapy for the maintenance of colitis patients. These data suggest that use of combination therapy is essential for the optimal inhibition of colitis-associated colorectal cancer.

Detection of colorectal adenomas using a bioactivatable probe specific for matrix metalloproteinase activity.

A significant proportion of colorectal adenomas, in particular those that lack an elevated growth component, continue to escape detection during endoscopic surveillance. Elevation of the activity of matrix metalloproteinases (MMPs), a large family of zinc endopeptidases, in adenomas serves as a biomarker of early tumorigenesis. The goal of this study was to assess the feasibility of using a newly developed near-infrared bioactivatable probe (MMPSense 680) that reports the activity of a broad array of MMP isoforms to detect early colorectal adenomas. Adenomatous polyposis coli (Apc)(+/Min-FCCC) mice that spontaneously develop multiple colorectal adenomas were injected with MMPSense 680, and the colons were imaged in an IVIS Spectrum system ex vivo. Image analyses were correlated with histopathologic findings for all regions of interest (ROIs). The biochemical basis of fluorescent signal was investigated by immunohistochemical staining of MMP-7 and -9. A strong correlation (Kendall = 0.80) was observed between a positive signal and the presence of pathologically confirmed colonic adenomas; 92.9% of the 350 ROIs evaluated were classified correctly. The correlation between two independent observers was 0.87. MMP-7 expression was localized to epithelial cells of adenomas and microadenomas, whereas staining of MMP-9 was found in infiltrating polymorphonuclear leukocytes within the adenomas. MMPSense 680 identifies colorectal adenomas, both polypoid and nonpolypoid, in Apc(+/Min-FCCC) mice with high specificity. Use of this fluorescent probe in combination with colonoscopy could aid in preventing colorectal neoplasias by providing new opportunities for early detection and therapeutic intervention.

Functional characterization of peroxisome proliferator-activated receptor-ß/δ expression in colon cancer.

This study critically examined the role of PPARß/δ in colon cancer models. Expression of PPARß/δ mRNA and protein was lower and expression of CYCLIN D1 protein higher in human colon adenocarcinomas compared to matched non-transformed tissue. Similar results were observed in colon tumors from Apc(+/Min-FCCC) mice compared to control tissue. Dietary administration of sulindac to Apc(+/Min-FCCC) mice had no influence on expression of PPARß/δ in normal colon tissue or colon tumors. Cleaved poly (ADP-ribose) polymerase (PARP) was either increased or unchanged, while expression of 14-3-3ε was not influenced in human colon cancer cell lines cultured with the PPARß/δ ligand GW0742 under conditions known to increase apoptosis. While DLD1 cells exhibited fewer early apoptotic cells after ligand activation of PPARß/δ following treatment with hydrogen peroxide, this change was associated with an increase in late apoptotic/necrotic cells, but not an increase in viable cells. Stable over-expression of PPARß/δ in human colon cancer cell lines enhanced ligand activation of PPARß/δ and inhibition of clonogenicity in HT29 cells. These studies are the most quantitative to date to demonstrate that expression of PPARß/δ is lower in human and Apc(+/Min-FCCC) mouse colon tumors than in corresponding normal tissue, consistent with the finding that increasing expression and activation of PPARß/δ in human colon cancer cell lines inhibits clonogenicity. Because ligand-induced attenuation of early apoptosis can be associated with more late, apoptotic/necrotic cells, but not more viable cells, these studies illustrate why more comprehensive analysis of PPARß/δ-dependent modulation of apoptosis is required in the future.

Endoscopic imaging and size estimation of colorectal adenomas in the multiple intestinal neoplasia mouse.

BACKGROUND: The scientific potential of animal models of carcinogenesis has not been fully realized because of our limited ability to monitor tumor growth in vivo. OBJECTIVE: To develop an endoscopy-based protocol for the accurate estimation of adenoma size in vivo from images obtained during colonoscopy. DESIGN: To compare estimates of lesion size acquired during endoscopy with those obtained from magnetic resonance imaging (MRI) and at necropsy. SETTING: A small-animal imaging facility. SUBJECTS: Adenomatous polyposis coli multiple intestinal metaplasia Fox Chase Cancer Center mice that develop multiple colorectal adenomas. METHODS: The mice received colonoscopic examination by using a rigid endoscope, and high-resolution images of colon adenomas were captured by using a charge-coupled-device camera. Lesion size was estimated by comparing the dimensions of the adenoma relative to a reference rod by using a novel geometric construction. The resulting areas were compared with estimates from MRIs and validated at necropsy. MAIN OUTCOME MEASUREMENTS: Cross-sectional area of colon adenomas. RESULTS: The cross-sectional area of 20 adenomas was measured in vivo during colonoscopy and compared with the size as measured at necropsy, which yielded a Pearson correlation coefficient of 0.94 (P = 6.52 x 10(-9)). Assessment of interoperator variability, when using measurements from 11 adenomas, yielded a Pearson correlation coefficient of 0.85 (P = 4.35 x 10(-3)) and demonstrated excellent reproducibility. LIMITATIONS: Only the distal colon could be viewed, and endoscopic measurements were 2-dimensional. CONCLUSIONS: An endoscopic method for the reliable measurement of colorectal adenomas in vivo was established. The application of this technique to mouse models of colon carcinogenesis will provide unique insight into the dynamics of adenoma growth.

5-aminosalicylic acid inhibits colitis-associated colorectal dysplasias in the mouse model of azoxymethane/dextran sulfate sodium-induced colitis.

BACKGROUND: The impact of the antiinflammatory agent 5-aminosalicylic acid (5-ASA) on the risk for colitis-associated colorectal cancer remains controversial. The chemopreventive activity of 5-ASA was evaluated in the Swiss Webster model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated neoplasia. METHODS: Mice were injected with AOM (7.4 mg/kg i.p.) and randomized to receive either vehicle or 5-ASA (75, 150, and 225 mg/kg) for the remainder of the study. DSS treatment began at 9 weeks of age and continued for 3 cycles. At the time of sacrifice (18 weeks of age), the entire colon and rectum were processed for histopathologic examination. RESULTS: An inverse trend was observed between dose and multiplicity of colonic dysplasias in all drug-treated groups (P = 0.03), with animals receiving 75 mg/kg 5-ASA exhibiting 56% of the number of dysplasias of the AOM/DSS controls (mean +/- SEM: 7.6 +/- 1.4 and 13.6 +/- 2.7, respectively). Administration of 75 mg/kg 5-ASA decreased both the mean multiplicity of flat dysplasias (1.8 +/- 0.4 for drug-treated versus 5.6 +/- 1.2 for AOM/DSS control) and the burden of polypoid dysplasias (tumor burden: 6.7 +/- 2.7 for drug-treated versus 14.9 +/- 3.9 units for AOM/DSS controls) significantly (P = 0.002 and 0.04, respectively). Inflammation was least severe in the 75 mg/kg group, which exhibited the fewest number of colorectal tumors. CONCLUSIONS: These data suggest that low-dose 5-ASA may be efficacious in preventing colitis-associated dysplasias and provide strong support for optimizing this therapy for the prevention of colonic neoplasms in patients with ulcerative colitis.

Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium.

Loss of p53 function is an early event in colitis-associated neoplasia in humans. We assessed the role of p53 in a mouse model of colitis-associated neoplasia. Colitis was induced in p53-/-, p53+/- and p53+/+ mice using three or four cycles of dextran sulfate sodium (DSS) followed by 120 days of water. Mice were examined for incidence, multiplicity and types of neoplastic lesions. Lesions were examined for mutations in beta-catenin (exon 3), K-ras (codons 12/13) and p53 (exons 5-8) by sequencing and for cellular localization of beta-catenin by immunohistochemistry. The incidence of neoplastic lesions was 57, 20 and 20% in p53-/-, p53+/- and p53+/+ mice, respectively (P = 0.013). p53-/- mice had a greater number of total lesions (P < 0.0001), cancers (P = 0.001) and dysplasias (P = 0.009) per mouse than either p53+/- or p53+/+ mice. Flat lesions were associated with the p53-/- genotype, whereas polypoid lesions were associated with the p53+/- and p53+/+ genotypes (P < 0.0001). beta-Catenin mutations were present in 75% of lesions of p53+/+ mice and absent in lesions from p53-/- mice (P = 0.055). Nuclear expression of beta-catenin was seen only in polypoid lesions (91%). No K-ras or p53 mutations were detected. These data indicate that loss of p53 enhances the induction of colitis-associated neoplasia, particularly flat lesions, and dysregulation of beta-catenin signaling plays an important role in the formation of polypoid lesions in this mouse model. As observed in humans, p53 plays a protective role in colitis-associated neoplasia in the DSS model.

Sulindac sulfone is most effective in modulating beta-catenin-mediated transcription in cells with mutant APC.

Sulindac sulfone (FGN-1, Aptosyn), a metabolite of the nonsteroidal anti-inflammatory drug sulindac, lacks cyclooxygenase inhibitory activity. Although its ability to inhibit tumorigenesis in both carcinogen-treated animals and patients with familial adenomatous polyposis has been attributed to the induction of apoptosis, its complete mechanism of action remains unclear. The purpose of the present study was to determine the ability of sulindac metabolites to regulate cellular levels of beta-catenin and downstream targets of the adenomatous polyposis coli (APC)/beta-catenin pathway in vitro. Sulindac sulfone was consistently more potent than the sulfide metabolite in all analyses, significantly decreasing the expression of total cellular beta-catenin (50% of control), pro-caspase 3 (49%), cyclin D1 (51%), and PPARdelta (65%) in SW480 cells. No significant alteration in pro-caspase 3 or beta-catenin expression was found in HCA7, LS174, or Caco-2 cells treated with sulindac sulfone. A dose-dependent reduction in TCF-mediated transcriptional activity was also observed in SW480 cells. These data demonstrate that sulindac sulfone can modulate the APC/beta-catenin pathway in vitro and that its efficacy is dependent upon the mutational status of APC and beta-catenin.

Generation of a unique strain of multiple intestinal neoplasia (Apc(+/Min-FCCC)) mice with significantly increased numbers of colorectal adenomas.

The relevance of the Apc(+/Min) mouse model in the study of human colorectal cancer remains uncertain due to the predominance of small intestinal adenomas and few, if any, colorectal adenomas. A new strain of Apc(+/Min) mice (Apc(+/Min-FCCC)) with significantly greater numbers of colorectal adenomas has been generated and characterized. Male C57BL/6J-Apc(+/Min) mice (the Jackson Laboratory, Bar Harbor, ME) were crossed with wild-type (Apc(+/+)) C57BL/6J females from an independent colony at this institution (offspring=Apc(+/Min-FCCC)) and 233 animals were evaluated over 20 generations. In order to determine the contribution of genetics to the enhanced colorectal adenoma phenotype, breeding pairs (Apc(+/Min) male x wild type female C57BL/6J) were purchased from the Jackson Laboratory and offspring (Apc(+/Min-JAX)) were maintained in our facility under identical conditions (n=98). Animals were fed Purina Rodent chow (#5013) diet containing 5% fat. The entire intestinal tract was examined histopathologically in both strains. Both the Apc and Pla2g2a (candidate for Mom1) genes were sequenced and found to be identical for both the Apc(+/Min-FCCC) and Apc(+/Min-JAX) mouse strains. The multiplicity of colorectal adenomas in the Apc(+/Min-FCCC) mice was much higher than reported in the literature and significantly greater than the multiplicity of colorectal adenomas in Apc(+/Min-JAX) mice maintained in our facility (P=0.01). Apc(+/Min-FCCC) had a significantly greater incidence of rectal prolapse (P = 0.02) and small intestinal adenocarcinomes (P=0.001), and multiplicity of small intestinal adenocarcinomas (P=0.001) compared to Apc(+/Min-JAX) mice. Male Apc(+/Min-FCCC) mice had significantly greater numbers of colorectal adenomas compared to female Apc(+/Min-FCCC) mice (P=0.0002), as did male Apc(+/Min-JAX) mice vs. female Apc(+/Min-JAX) mice (P< 0.0001). These results allow us to conclude: (1) Apc(+/Min-FCCC) mice are unique in that they develop significantly greater numbers of colorectal adenomas and small intestinal cancers, and a significantly greater incidence of small intestinal cancers and rectal prolapse than Apc(+/Min-JAX) mice. (2) This study represents the first report of a significant gender difference in multiplicity of colorectal adenomas. (3) Differences between Apc(+/Min-FCCC) and Apc(+/Min-JAX) mice in currently undefined genetic modifiers may contribute to the enhanced colorectal phenotype. (4) The Apc(+/Min-FCCC) strain is highly suited for the investigation of colorectal neoplastic disease and chemoprevention studies.

beta-catenin-mediated signaling: a molecular target for early chemopreventive intervention.

Dysregulation of Wnt signaling appears to be a critical event in the formation of intestinal tumors and some other cancers. Accumulating data from preclinical studies strongly suggest that targeted disruption of beta-catenin-mediated TCF signaling is a promising strategy for early chemopreventive intervention, particularly with respect to intestinal tumorigenesis. While the search for potent inhibitors is just getting underway, the ability of several synthetic and naturally occurring agents to decrease the transcriptional activity of a luciferase reporter plasmid under the control of TCF-4 regulatory elements (pTOPFLASH) has been demonstrated already. Additional enthusiasm for this approach is provided by data from several groups, which indicate that sulindac, sulindac sulfone and indomethacin can modulate the subcellular localization of beta-catenin in vivo, resulting in either decreased nuclear compartmentalization or enhanced localization of beta-catenin to the plasma membrane. Although the mechanism by which agents disrupt beta-catenin-mediated TCF signaling remains to be elucidated, possibilities include: (1) physical inhibition of the beta-catenin/TCF complex formation, (2) upregulation of the ubiquitin-mediated proteosomal degradation of beta-catenin, (3) accelerated nuclear export of beta-catenin and (4) enhanced sequestration of beta-catenin by E-cadherin. The common role of beta-catenin in both Wnt signaling and cell adhesion provides a unique opportunity to develop chemopreventive therapies that both prevent the development of cancer and delay tumor progression.

Perillyl alcohol-mediated inhibition of lung cancer cell line proliferation: potential mechanisms for its chemotherapeutic effects.

Perillyl alcohol (POH) is currently being tested in clinical trials as an anticancer agent, though its mechanism of action has not been definitively established. We treated two human lung cancer cell lines, H322 and H838, with POH to determine its antitumor properties. A sulforhodamine B (SRB) cell proliferation assay was used to determine the effects of POH after 1 and 5 days of treatment with 0.25, 0.5, 0.75, 1.0, and 1.5 mM POH. After 1 day of treatment, little difference could be seen between the lowest and highest concentrations of POH. However, after 5 days, both cell lines showed a dose-dependent decrease in cell proliferation that ranged from 15% to 83%. A clonogenic assay confirmed these results-while there was no significant effect of POH after 1 day of exposure, a dose-dependent decrease in colony formation, ranging from 15% to 100%, was seen after 5 days of treatment. Time-lapse video microscopy revealed that apoptotic cells were evident within 24-48 h of treatment with 1.5 mM POH. The appearance of apoptotic cells was preceded by increased caspase-3 activity and cleavage of poly (ADP-ribose) polymerase (PARP) as POH activated caspase-3 activity 3-6-fold. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) confirmed the classical characteristics of apoptosis in POH-treated cells. DNA microarray expression analysis was performed following 8 and 24 (H322) or 8 and 48 (H838) h of treatment with 1.5 mM POH. While a large number of genes were up- or downregulated in the two cell lines at various times after POH treatment, the levels of expression of only eight genes were up- or down-related in both cell lines at both of the time points examined. The significance of these genes as potential mediators of POH action is still uncertain, but the limited number of commonly up- or downregulated genes detected by microarray expression analysis suggests that POH may mediate its effects via posttranscriptional mechanisms. Our results suggest that POH may have potential use as an anticancer drug that stimulates or sensitizes lung tumor cells to apoptosis, and this effect may depend on genetic lesions present in tumor cells.