Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay.

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P-DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 microM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10(6) bp versus 3.7 lesions/10(6) bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3-10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mbeta16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P-DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.

Age-associated changes in the expression pattern of cyclooxygenase-2 and related apoptotic markers in the cancer susceptible region of rat prostate.

Senescence-associated changes in the prostate are believed to play an important role in the genesis of prostate cancer. In order to provide further information on how aging increases the prostate susceptibility to cancer, we examined the pattern of cyclooxygenase (COX)-2 expression and the concomitant alterations in prostaglandin E(2) (PGE(2)) synthesis in the prostate glands of 4-, 10-, 50- and 100-week-old Fischer 344 rats. This was carried out in the prostatic areas where hormone-induced tumors arise, namely the periurethral ducts of the dorsolateral prostate (DLP). Age-associated changes were also evaluated for pro- and anti-apoptotic factors linked to COX-2 signaling and known to be involved in the normal development of the prostate gland as well as in carcinogenesis. COX-2 expression was increased in the DLP in an age-dependent manner where senescent rats had >3-4-fold higher COX-2 mRNA and protein levels than their juvenile counterparts (P<0.05). The age-related changes in COX-2 were accompanied by a similar up-regulation in the PGE(2) synthesis. Evaluation of mediators of apoptotic signaling showed a significant (P<0.05) decline in the expression levels of the pro-apoptotic BAX (>6-fold) and peroxisome proliferator-activated receptor gamma (>3-fold) and in caspase-3 activity (>2-fold) and an up-regulation of the anti-apoptotic Bcl(2) (>8-fold), PKCalpha (>2-fold) and pAkt (>4-fold) in the 100-week-old rats versus the 4-week-old animals. There was an approximately 15-fold age-dependent decrease in the pro-apoptotic ratio BAX:Bcl(2) and an increase in the anti-apoptotic variable PKCalpha(*)Bcl(2)/BAX in the senescent rats compared with the juvenile ones. These results suggest that increased COX-2 expression can be linked to the decline in the pro-apoptotic signaling in the prostate gland during aging. Subsequently, COX-2 inhibitors can be considered as a promising class of agents to attenuate the increased cell survival and, hence, protect against tumorigenesis in the aging prostate.

Inhibition of rat mammary gland carcinogenesis by simultaneous targeting of cyclooxygenase-2 and peroxisome proliferator-activated receptor gamma.

We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, and N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu), a peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-L-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treatments (P < 0.05). The effects of the test drugs on COX-2 and PPAR gamma expression and on the synthesis of prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were examined in rat mammary normal (MNU-untreated), uninvolved, and tumor (MNU-treated) tissues. Celecoxib and F-L-Leu, separately, inhibited COX-2 and up-regulated PPAR gamma expression. These effects were paralleled by inhibition of PGE(2) synthesis and up-regulation of 15d-PGJ(2). Combined treatment resulted in higher alterations in COX-2 and PPAR gamma transcripts and PG synthesis compared with separate administrations. The effect of the test agents on Bcl(2), BAX, and protein kinase C alpha expression levels were examined in the rat mammary gland and the pro-(BAX:Bcl(2)) and anti-[PKC alpha*(Bcl(2)/BAX)] apoptotic ratios were evaluated. Each drug increased the proapoptotic ratio by 2- to 7-fold and reduced the antiapoptotic ratio by 2- to >8-fold in all tissues. Combined treatment, however, resulted in >9- to 14-fold up-regulation in the proapoptotic processes and 15- to >30-fold down-regulation in the antiapoptotic ones. Analyses were also carried out on the drug-induced modulation of cell cycle regulators and proliferation markers (cyclin-dependent kinase 1 and proliferating cell nuclear antigen). F-L-Leu and celecoxib each reduced the cyclin-dependent kinase 1 and proliferating cell nuclear antigen expression in the tumor. Higher down-regulation was attained in all tissues by combined treatment where cyclin-dependent kinase 1 and proliferating cell nuclear antigen almost retained the expression levels observed in the normal glands. In conclusion, simultaneous targeting of COX-2 and PPAR gamma may inhibit mammary cancer development more effectively than targeting each molecule alone. COX-2 inhibitors and PPAR gamma agonists coordinately mediate their anticancer effect via both COX-dependent (inhibition of COX-2, activation of PPAR gamma, and modulation PG synthesis) and COX-independent (induction of proapoptotic factors and inhibition of cell proliferation) pathways.

Relative imbalances in estrogen metabolism and conjugation in breast tissue of women with carcinoma: potential biomarkers of susceptibility to cancer.

Exposure to estrogens has been associated with an increased risk of developing breast cancer. Breast biopsy tissues from 49 women without breast cancer (controls) and 28 with breast carcinoma (cases) were analyzed by HPLC with electrochemical detection for 31 estrogen metabolites and catechol estrogen quinone-glutathione conjugates. The levels of estrone and estradiol were higher in cases. More 2-catechol estrogen (CE) than 4-CE was observed in controls, but the 4-CE were three times higher than 2-CE in cases. In addition, the 4-CE were nearly four times higher in cases than in controls. Less O-methylation was observed for the CE in cases. The level of catechol estrogen quinone conjugates in cases was three times that in controls, suggesting in the cases a higher probability for the quinones to react with DNA and generate mutations that may initiate cancer. The levels of 4-CE and quinone conjugates were highly significant predictors of breast cancer. These results suggest that some catechol estrogen metabolites and conjugates could serve as biomarkers to predict risk of breast cancer.

Inhibition of cyclooxygenase-2 and activation of peroxisome proliferator-activated receptor-gamma synergistically induces apoptosis and inhibits growth of human breast cancer cells.

Cyclooxygenase-2 (COX-2) expression and peroxisome proliferator-activated receptor-gamma (PPARgamma) inactivation are linked to increased risk of human breast cancer. This study examines the effect of simultaneous targeting of COX-2 and PPARgamma on the proliferation of human breast cancer cells and on the expression of Bcl-2, BAX, and caspases-3 and -9, modulators of apoptotic cell death. Treatment of MDA-MB-231 breast cancer cells with NS-398 (a COX-2 inhibitor) or ciglitazone (CGZ, a PPARgamma-ligand) significantly inhibited cell proliferation and markedly increased apoptotic rates. These effects were accompanied by upregulation of BAX and caspases-3 and -9 mRNA expression and downregulation of Bcl-2. Compared to the influence of separate treatments, simultaneous treatment with NS-398 and CGZ synergistically inhibited cell proliferation and induced apoptotic cell death. In conclusion, combinational targeting of COX-2 and PPARgamma can inhibit the growth of human breast cancer cells and induce apoptosis to an extent more suprior to that produced by targeting each molecule alone. COX-2 and PPARgamma can be promising molecular targets for combinational chemoprevention or treatment of breast cancer.

Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma and levels of prostaglandin E2 and 15-deoxy-delta12,14-prostaglandin J2 in human breast cancer and metastasis.

Cyclooxygenase-2 (COX-2) expression and peroxisome proliferator-activated receptor-gamma (PPARgamma) inactivation are linked to increased risk of human breast cancer. The purpose of our study was to examine the relationship between COX-2 (with the resulting prostaglandins E(2), PGE(2)) and PPARgamma (and its natural endogenous ligand 15-Deoxy-Delta(12,14)-prostaglandin J(2), 15d-PGJ(2)) at various stages during the development of human breast cancer and its progression to metastasis. Human breast tissue specimens were collected from normal breasts or from individuals with fibrocystic disease and served as controls (n = 22). Tissues were also collected from uninvolved (n = 25), tumor (n = 25) and lymph node metastasis (n = 15) regions from breast cancer patients. COX-2 and PPARgamma mRNA expression were increased and downregulated, respectively, in tissues from cancer patients compared to controls. Metastatic tissues tended to have higher alterations compared to non-metastatic tissues (p < 0.05). These altered expressions in COX-2 and PPARgamma were paralleled by increases in the tissue levels of PGE(2) and decreases in 15d-PGJ(2). A significant inverse correlation was found between PGE(2) and 15-d-PGJ(2) (r = -0.51, p < 0.05). Significant correlations (p < 0.05) were also obtained between COX-2 and PPARgamma mRNA (inverse, r = -0.72) and between COX-2 and PGE(2) (direct, r = 0.68). Increases in COX-2 mRNA expression and levels of PGE(2) and down-regulation of PPARgamma mRNA expression and 15d-PGJ(2) levels were characterized as predictors of breast cancer risk (p < 0.05). Our results suggest that the altered expression of COX-2 and PPARgamma and the subsequent modulation in the tissue levels of PGE(2) and 15-d-PGJ(2) may influence the development of human breast cancer and its progression to metastasis.

DNA alkylation and repair in the large bowel: animal and human studies.

O6-methylguanine (O6-MeG), a procarcinogenic DNA adduct that arises from exposure to methylating agents, has been detected in human colorectal DNA at levels comparable to those that cause adverse effects in model systems. O6-MeG levels vary within the colon, being higher in the cancer-prone regions of the large bowel. In rats and mice, O6-MeG persistence in colon DNA is associated with the induction of colon tumors after treatment with methylating agents. These tumors frequently contain K-ras GC-->AT transition mutations, which is consistent with the mutagenic properties of O6-MeG: such mutations are also commonly found in human colorectal cancers. O6-Alkylguanine adducts are removed by the DNA repair protein, O6-alkylguanine DNA-alkyltransferase (MGMT). MGMT overexpression in transgenic mice reduces the formation of K-ras GC-->AT mutations and tumors induced by methylating agents. Interindividual variations in human colon MGMT activity are large and large bowel tumors can occur in regions of low activity. Low MGMT activity in normal mucosa has been associated with the occurrence of K-ras GC-->AT mutations, whereas reduced MGMT expression and an increased frequency of K-ras GC-->AT mutations in colorectal cancers have been linked to MGMT promoter methylation. MGMT activity is also lower in adenomas than in adjacent normal tissue but only in those adenomas with this specific mutation. These results are entirely consistent with the hypothesis that GC-->AT mutations in the K-ras oncogene result from the formation and persistence of O6-alkylguanine lesions in colorectal DNA. Human exposure to endogenous or exogenous alkylating agents may thus be an environmental determinant of colorectal cancer risk.

Influence of cigarette smoking on prostaglandin synthesis and cyclooxygenase-2 gene expression in human urinary bladder cancer.

The present study examines the effect of tobacco smoking on the expression of cyclooxygenase (COX)-2 gene, COX enzymatic activity and prostaglandin (PG) synthesis in urothelial mucosal tissues from patients with bladder cancer and from normal individuals. The detection frequency of COX-2 mRNA was 2-fold higher in bladder cancer patients compared to controls and it was accompanied by a significantly increased COX enzymatic activity and PGE2 synthesis (p < 0.05). Smokers, in both control and patients groups, had higher COX-2 expression, COX activity, and PGE2 synthesis compared to the nonsmokers (p < 0.05). The number of cigarettes smoked in the cases, but not controls, correlated well with COX enzymatic activity (r = 0.42, p = 0.016). The observed over-expression of COX-2 gene in human urinary bladder and the concomitant increases in PG synthesis may explain, at least in part, the mechanism by which cigarette smoking influences the development of urothelial neoplasia.

Cyclooxygenase-2 inhibitor celecoxib inhibits promotion of mammary tumorigenesis in rats fed a high fat diet rich in n-6 polyunsaturated fatty acids.

The objective of this investigation was to determine whether celecoxib, a highly specific inhibitor of cyclooxygenase-2 (COX-2), inhibits the promotion phase of mammary tumorigenesis in rats fed a high fat diet rich in n-6 polyunsaturated fatty acids (PUFAs) that is known to induce COX-2 expression. Sixty female Sprague-Dawley rats were initially maintained on an AIN-93G diet. At 50 days of age they received a single i.p. injection of methylnitrosourea (MNU). One week later, all rats were switched to a modified AIN-93G diet containing 18% safflower oil plus 3% soybean oil. Half of the rats also began receiving 1500 ppm celecoxib in the diet and the control and experimental diets were continued for a further 23 weeks. Celecoxib significantly decreased both the final tumor incidence (63.3% in the celecoxib group versus 82.2% in the control group, P<0.05) and tumor multiplicity (0.9+/-0.2 tumors/rat in the celecoxib group versus 2.3+/-0.3 tumors/rat in the control group, P<0.05). At the termination of the experiment, body weights were significantly lower in the celecoxib group compared to controls (330.6+/-6.1 versus 401.5+/-10.9 g respectively, P<0.05) although there was no evidence of toxicity and food intakes were not different for the two groups. Fasting serum triglycerides and abdominal adipose tissue accumulation were lower in the celecoxib group compared to controls (49.3+/-4.4 versus 82.8+/-12.6 mg/dL, P<0.05, and 7.2+/-0.3 versus 11.3+/-0.4% of body weight, P<0.01, respectively). These results show that administration of celecoxib to rats in a high fat diet rich in n-6 PUFAs suppresses the promotion of mammary tumorigenesis induced by MNU. This inhibition may be due to the effects of celecoxib on lipid metabolism as well as COX-2.

Chemoprevention of breast cancer by targeting cyclooxygenase-2 and peroxisome proliferator-activated receptor-gamma (Review).

Cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor-gamma (PPARgamma) have emerged as candidate molecules that hold great promise for cancer chemoprevention. COX-2 increased expression and PPARgamma inactivation occur during mammary gland carcinogenesis. COX-2 and PPARgamma may contribute to breast cancer induction either directly or via their effects on factors known to influence tumor development, e.g., nuclear factor-kappaB and vascular endothelial growth factor. Inhibition of COX-2 or activation of PPARgamma prevents mammary carcinomas in experimental animals with little toxicity. Combinational treatment with COX-2 inhibitor and PPARgamma agonists may produce synergistic anti-tumorigenic effects without significant toxicity and, therefore, be an effective strategy to prevent human breast cancer. Establishing a relationship between COX-2 and PPARgamma in this malignancy may provide the basis for a novel chemopreventive strategy based on the modulation of both molecules simultaneously. This review evaluates experimental and epidemiological findings suggesting a possible role of COX-2 and PPARgamma in the development of human breast cancer and presents evidence substantiating their coordinated action in carcinogenesis and finally develops a rationale for the simultaneous targeting of both molecules as a potentially effective strategy to prevent breast malignancy.

Catechol estrogen metabolites and conjugates in different regions of the prostate of Noble rats treated with 4-hydroxyestradiol: implications for estrogen-induced initiation of prostate cancer.

Prostate carcinomas arise in 100% of Noble rats treated with estradiol and testosterone. We hypothesize that estrogens initiate prostate cancer mainly by formation of 4-catechol estrogens (CE), followed by their oxidation to catechol estrogen-3,4-quinones (CE-3,4-Q), which can react with DNA. To avoid cancer initiation, CE can be detoxified by catechol-O-methyltransferase (COMT), and CE-3,4-Q by conjugation with glutathione (GSH) or by reduction to CE, catalyzed by quinone reductase and/or cytochrome P450 reductase. To investigate the prostatic metabolism of estrogens, Noble rats were treated with the CE 4-hydroxyestradiol (4-OHE2) or estradiol-3,4-quinone (E2-3,4-Q), and CE metabolites and conjugates were analyzed in the four regions of the prostate, which differ in susceptibility to carcinoma formation. Following treatment of rats with 4-OHE2 (6 micromol/100 g body weight in 200 microl of trioctanoin/dimethylsulfoxide (4:1) by intraperitoneal injection) for 90 min, the non-susceptible ventral (VP) and anterior (AP) prostate had higher levels of 4-methoxyCE and GSH conjugates than the susceptible dorsolateral prostate (DLP) and periurethral prostate (PUP). After treatment with the same molar amount of E2-3,4-Q, the VP and AP contained more GSH conjugates, 4-CE and 4-methoxyCE than the susceptible DLP and PUP. These results suggest that prostate areas susceptible to carcinoma induction have less protection by COMT, GSH, and quinone reductase and/or cytochrome P450 reductase, favoring reaction of CE-3,4-Q with DNA, presumably to initiate cancer.