Exisulind in combination with celecoxib modulates epidermal growth factor receptor, cyclooxygenase-2, and cyclin D1 against prostate carcinogenesis: in vivo evidence.

PURPOSE: Nonsteroidal anti-inflammatory drugs mediate anticancer effects by modulating cyclooxygenase-2 (COX-2)-dependent and/or COX-2-independent mechanism(s); however, the toxicity issue is a concern with single agents at higher doses. In this study, we determined the combined effect of celecoxib, a COX-2 inhibitor, along with exisulind (sulindac sulfone/Aptosyn) at low doses in prostate cancer. EXPERIMENTAL DESIGN: We used a sequential regimen of N-methyl-N-nitrosourea + testosterone to induce prostate cancer in Wistar-Unilever rats. Following carcinogen treatment, celecoxib and exisulind individually and their combination at low doses were given in NIH-07 diet for 52 weeks. We determined the incidence of prostatic intraepithelial neoplasia, adenocarcinomas, rate of tumor cell proliferation, and apoptosis. Immunohistochemical and Western blot analysis were done to determine COX-2, epidermal growth factor receptor (EGFR), Akt, androgen receptor, and cyclin D1 expression. Serum prostaglandin E2 and tumor necrosis factor-alpha levels were determined using enzyme immunoassay/ELISA assays. RESULTS: The rats that received celecoxib in combination with exisulind at low doses showed a significant decrease in prostatic intraepithelial neoplasia and adenocarcinomas as well as an enhanced rate of apoptosis. An overall decrease in COX-2, EGFR, Akt, androgen receptor, and cyclin D1 expression was found associated with tumor growth inhibition. Reduced serum levels of COX-2 protein, prostaglandin E2, and tumor necrosis factor-alpha indicated anti-inflammatory effects. A strong inhibition of total and phosphorylated form of EGFR (Tyr(992) and Tyr(845)) and Akt (Ser(473)) was significant in rats given with these agents in combination. CONCLUSIONS: In this study, we show for the first time that the combination of celecoxib with exisulind at low doses could prevent prostate carcinogenesis by altering key molecular events.     

Papilloma and carcinoma production in DMBA-initiated, onion oil-promoted mouse skin

Groups of 20 females Ha/ICR mice were initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene (DMBA) and promoted one week later with topical treatments three times per week of 5 micrograms phorbol myristate acetate (PMA) and/or onion oil or garlic oil. Promotion was continued for 49 weeks in most experiments. Promotion was continued for 60 weeks in the experiment that evaluated the effect of time intervals between PMA and garlic oil. All experiments were conducted with 0.2 ml acetone solutions of agents. Onion oil, but not garlic oil, was a weak promoter in mouse skin. A 1-mg dose produced five papillomas in three mice and one carcinoma in 330 days (18 survivors). The 10-mg dose was more effective; it produced cumulative yields of 56 papillomas in 14 mice and 7 carcinomas in 4 mice in 345 days (14 survivors). Onion oil is neither an initiator nor a whole carcinogen. The effects of intervals between PMA and a 1-mg dose of onion or garlic oil were determined. These intervals were -2 hrs, -1 hr, -0.5 hr, +0.5 hr, +1 hr, and +2 hrs with respect to time of PMA application. Maximal inhibition of papillomas by onion oil was observed at the +0.5-hr interval and was similar to that previously reported. Garlic oil is not a promoter. It inhibited papillomas at the +0.5-hr, +1.0-hr, and +2.0-hr intervals but did not appear to affect carcinoma production.     

Frequent activation of the Ki-ras oncogene at codon 12 in N-methyl-N-nitrosourea-induced rat prostate adenocarcinomas and neurogenic sarcomas.

Rat neoplasms induced by methylating carcinogens frequently contain ras genes activated by a single point mutation. Rat prostatic tumors induced by a combination of a single injection of N-methyl-N-nitrosourea (MNU) and long-term treatment with testosterone were examined for the presence of such activating point mutations in ras genes. These tumors, which arose exclusively in the dorsolateral prostate, included both adenocarcinomas and sarcomas. Activating mutations in codon 12 of the Ki-ras gene were found in 7 of 10 carcinomas and 4 of 5 sarcomas, using selective oligonucleotide hybridization analysis of DNA amplified by the polymerase chain reaction (PCR). However, no mutated Ha-ras oncogenes were detected. The presence of PCR-engineered Hphl restriction sites created by the existence of a G35----A mutation in the rat Ki-ras oncogene identified the mutation as a GC----AT transition at the second position of codon 12. Production of O6-methylguanine adducts in the Ki-ras codon 12 followed by base mispairing during replicative DNA synthesis is thus the likely molecular mechanism of initiation of prostatic carcinogenesis by MNU in the rat. Three of the four sarcomas positive for the Ki-ras G35----A mutation were immunohistochemically defined as of Schwann cell origin, indicating that involvement of the ras gene family is possible in tumorigenesis of this cell lineage. Loss of the wild-type Ki-ras allele was also observed in all four of these sarcomas.     

18α-glycyrrhetinic acid targets prostate cancer cells by down-regulating inflammation-related genes

Glycyrrhetinic acid is an active triterpenoid metabolite of glycyrrhizin abundantly present in licorice roots. Glycyrrhetinic acid exists as α and β stereo-isomeric forms. Both stereo-isomeric forms are known to have anti-inflammatory and anticancer activity. However, the effects and anticancer mechanism of α glycyrrhetinic acid in prostate cancer cells has not yet been evaluated. Therefore, we investigated the growth inhibition, induction of apoptosis and the anticancer mechanisms of 18α-glycyrrhetinic acid (AGA), on the androgen-independent metastatic prostate cancer cell line DU-145. Our results showed that AGA inhibited proliferation and growth of these cells by inducing apoptosis as determined by Annexin V and flow cytometry analyses. Our studies also showed that HUVEC tube formation was drastically reduced when cultured in conditioned medium of AGA-treated DU-145 cells. In addition, AGA treatment prevented the invasion of DU-145 prostate cancer cells on matrigel coated transwells via down-regulation of NF-κB (p65), VEGF and MMP-9 expression. Furthermore, AGA treatment also down-regulated the expression of pro-inflammatory cytokine/growth factor genes HMGB1, IL-6 and IL-8 in DU-145 cells. Interestingly, AGA simultaneously upregulated the expression of non-steroidal anti-inflammatory gene-1 (NAG-1) in DU-145 cells suggesting its anti-inflammatory activity on prostate cancer cells. Taken together, the results of this study suggest that AGA may be a promising anticancer agent that merits further investigation for the chemoprevention and treatment of prostate cancer.     

Induction of proliferative lesions of ventral prostate, seminal vesicle, and other accessory sex glands in rats by N-methyl-N-nitrosourea: effect of castration, pretreatment with cyproterone acetate and testosterone propionate and rat strain.

Wistar (Cpb:WU), F344 or Sprague-Dawley rats were sequentially treated with cyproterone acetate (CA) for 21 days, testosterone propionate (TP) for 3 days, followed by a single i.v. injection of N-methyl-N-nitrosourea (MNU). One group of Wistar rats was castrated 4 weeks after MNU injection, and another group 58 weeks after MNU, when the first prostatic carcinoma was detected. Control groups received only CA + TP, CA, MNU, or they remained untreated. Early or late castration inhibited the development of atypical hyperplasia of the ventral prostate in Wistar rats. This lesion was induced by the CA + TP + MNU treatment in F344 rats, but not Sprague-Dawley rats; in Wistar rats, it was induced by CA + TP treatment, irrespective of whether MNU was given. Hypertrophic-hyperplastic lesions of the seminal vesicle were induced by MNU, irrespective of pretreatment, and their development was prevented by early castration and inhibited by late orchiectomy. Dorsolateral prostate carcinomas and preneoplasia occurred only in low incidence in Wistar and Sprague-Dawley rats. These lesions were absent in F344 rats that had received treatment with CA + TP + MNU. No dorsolateral prostate (pre)neoplasia was found in Wistar rats subjected to early orchiectomy, but rats castrated at 58 weeks had an incidence similar to that for the intact group treated with CA + TP + MNU. This finding supports the contention that androgens are required for the development of MNU-induced prostatic cancer in rats but that advanced carcinomas are androgen insensitive. Differences in incidence and localization of prostatic proliferative lesions between F344 and Wistar rats and between dorsolateral and ventral prostate could not be explained by differences in epithelial cell proliferative responses to CA + TP treatment at the time of MNU injection, since they were similar in ventral and dorsolateral prostate and were more prominent in F344 rats than in Wistar rats. DNA damage as estimated by MNU-induced unscheduled DNA synthesis also did not differ between dorsolateral and ventral prostate.     

Docosahexaenoic acid in combination with celecoxib modulates HSP70 and p53 proteins in prostate cancer cells

The role of cyclooxygenase-2 (COX-2) and the mechanism by which it influences the development and behavior of prostate cancer is unclear. Selective COX-2 inhibitors may be effective against prostate cancer via COX-2-independent mechanisms. But administration of high doses of COX-2 inhibitors over longer period of time may not be devoid of side effects. There is increasing interest in using COX-2 inhibitors in combination with other chemopreventive agents to overcome the issue of toxicity. However, the molecular mechanisms underlying their combined actions are not well understood. Therefore, the present study was designed to determine the effects of low doses of docosahexaenoic acid (DHA) in combination with celecoxib on the molecular targets at the proteins level in rat prostate cancer cells. Two-dimensional gel electrophoresis, in combination with mass spectrometry analysis, was used for protein identification. Western blot analysis confirmed the proteins identified. Paraffin-embedded tissue sections from the rat prostate tumor were used to detect base level expression of heat shock protein 70 (HSP70) and p53. The rate of cancer cell growth was inhibited more effectively (p < 0.01) by DHA in combination with celecoxib at lower doses (2.5 microM each). A total number of twelve proteins were differentially expressed by the combined action of DHA and celecoxib at low doses. It was interesting to note that these agents activated both HSP70 and p53 proteins. Activation of HSP70 by the combined actions of DHA and celecoxib in the presence of wild-type p53 reveals a unique COX-2 independent mode of action against prostate cancer.     

Cancer chemoprevention research with selenium in the post-SELECT era: Promises and challenges

The negative efficacy outcomes of double-blinded, randomized, placebo-controlled Phase III human clinical trials with selenomethionine (SeMet) and SeMet-rich selenized-yeast (Se-yeast) for prostate cancer prevention and Se-yeast for prevention of nonsmall cell lung cancer (NSCLC) in North America lead to rejection of SeMet/Se-yeast for cancer prevention in Se-adequate populations. We identify 2 major lessons from the outcomes of these trials: 1) the antioxidant hypothesis was tested in wrong subjects or patient populations, and 2) the selection of Se agents was not supported by cell culture and preclinical animal efficacy data as is common in drug development. We propose that next-generation forms of Se (next-gen Se), such as methylselenol precursors, offer biologically appropriate approaches for cancer chemoprevention but these are faced with formidable challenges. Solid mechanism-based preclinical efficacy assessments and comprehensive safety studies with next-gen Se will be essential to revitalize the idea of cancer chemoprevention with Se in the post-SELECT era. We advocate smaller mechanism-driven Phase I/II trials with these next-gen Se to guide and justify future decisions for definitive Phase III chemoprevention efficacy trials.     

Use of animal models in defining efficacy of chemoprevention agents against prostate cancer.

Animal models are crucial in preclinical efficacy testing of chemoprevention agents. The most feasible, realistic, and potentially effective target for prostate cancer chemoprevention is progression from prostatic intraepithelial neoplasia (PIN) to histologic cancer and from histologic to clinically manifest cancer. There are transgenic mouse models for prostate cancer and models for PIN, but these have not yet been fully developed and evaluated for chemoprevention studies. Human prostate cancer xenografts in mice and transplantable Dunning rat prostate carcinomas can be used to assess tumor growth inhibition. Several Dunning tumors metastasize, enabling detection of inhibition of metastases. Detection of inhibitory effects on de novo prostate cancer development requires induction of a high cancer incidence and similarity of induced tumors to human prostate carcinomas. Transgenic mice with oncogenes expressed in a prostate-specific fashion, combined chronic treatment of NBL rats with estradiol-17beta and testosterone, and sequential treatment of rats with carcinogens such as N-methyl-N-nitrosourea (MNU) and chronic testosterone treatment all lead to a high incidence of prostatic adenocarcinomas. PIN occurs mostly in the former two models, and metastases are frequent in some transgenic models and the MNU-testosterone rat model. The latter model has been applied to chemoprevention agent efficacy testing. In 8 control groups, the carcinoma incidence was 77% in all accessory sex glands combined, 51% for small tumors confined to dorsolateral/anterior prostate, and 25% for large tumors of uncertain origin in the prostate area. This model was predictive of the lack of antiprostate cancer efficacy of N-(4-hydroxyphenyl)all-trans-retinamide in humans. Thus, rats given MNU and chronic testosterone represent a relevant and reliable model for efficacy testing of chemoprevention agents. In conclusion, there are now adequate animal models for prostate cancer proven to be suitable for preclinical chemoprevention studies.