PAX2 function, regulation and targeting in fallopian tube-derived high-grade serous ovarian cancer.

The fallopian tube epithelium (FTE) is one of the progenitor populations for high-grade serous ovarian cancer (HGSC). Loss of PAX2 is the earliest known molecular aberration in the FTE occurring in serous carcinogenesis followed by a mutation in p53. Pathological studies report consistent loss of PAX2 in benign lesions as well as serous tumors. In the current study, the combined loss of PAX2 and expression of the R273H p53 mutant protein in murine oviductal epithelial (MOE) cells enhanced proliferation and growth in soft agar in vitro but was insufficient to drive tumorigenesis in vivo. A serially passaged model was generated to investigate the role of aging, but was also insufficient to drive tumorigenesis. These models recapitulate early benign lesions and suggest that a latency period exists between loss of PAX2, p53 mutation and tumor formation. Stathmin and fut8 were identified as downstream targets regulated by loss of PAX2 and mutation of p53 in MOE cells. Re-expression of PAX2 in PAX2-null human HGSC cells reduced cell survival via apoptosis. Phosphatase and tensin homolog (PTEN)shRNA negatively regulated PAX2 expression and stable re-expression of PAX2 in MOE:PTENshRNA cells significantly reduced proliferation and peritoneal tumor formation in athymic nude mice. PAX2 was determined to be a direct transcriptional target that was activated by wild-type p53, whereas mutant p53 inhibited PAX2 transcription in MOE cells. A small molecule screen using the proximal PAX2 promoter driving luciferase identified four small molecules that were able to enhance PAX2 mRNA expression in MOE cells. PAX2 re-expression in HGSC cells and PTEN-deficient oviductal tumors may have the potential to induce apoptosis. In summary, mutant p53 and PTEN loss negatively regulated PAX2 and PAX2 re-expression in HGSC cells induced cell death.

Effect of deguelin on UVB-induced skin carcinogenesis.

Nonmelanoma skin cancer afflicts more than one million people in the U.S. annually, highlighting the need for more effective preventive regimens. We have investigated the ability of deguelin, a plant-derived rotenoid with cancer chemopreventive activity, to inhibit UVB-induced skin carcinogenesis with the SKh-1 mouse model. Topically-applied deguelin significantly inhibited the multiplicity of UVB-induced skin tumors, indicating potential as a human skin cancer chemopreventive agent. Mechanistic studies to determine the potential of deguelin to block a number of established UVB-induced molecular events yielded negative results [including UVB-induced AP-1 DNA binding, c-fos and TNFalpha mRNA induction, arachidonic acid release and UVB-induced phosphorylation of mTOR (Ser2448), akt (Ser473) and erk (Thr202/Tyr204)]. These results are of interest as they contradict a major hypothesis for the mode of action of deguelin, i.e., a general down regulation of signal transduction based on inhibition of NADH dehydrogenase and depletion of ATP levels. In the current work, however, deguelin was found to activate 5' AMP-activated kinase (AMPK), a protein that acts as a cellular energy sensor. This is the first report of a chemopreventive agent having this effect and suggests a possible role for AMPK in cancer chemoprevention.

Prostate intraepithelial neoplasia in Noble rats, a potential intermediate endpoint for chemoprevention studies.

In most prostate chemoprevention studies conducted with animal models, the incidence and multiplicity of tumours have been used as endpoints for efficacy. However, the latency of tumours is usually over 1 year, making these studies costly and time consuming. The main purpose of this study was to assess the utility of prostate intraepithelial neoplasia (PIN), induced in Noble rats by continuous testosterone + oestradiol (T + E) administration, as a potential intermediate endpoint biomarker of efficacy in chemoprevention studies. Noble rats at the age of 12 weeks were treated for 36 weeks with T + E given subcutaneously via Silastic capsules. The incidence and multiplicity of PIN were assessed in various prostate glands by serial sections generated at three separate tissue levels. The efficacy of dehydroepiandrosterone (DHEA) and DHEA 8354 (1000 and 2000 mg/kg diet), difluoromethylornithine (DFMO) (1000 and 2000 mg/kg diet) and oltipraz (125 and 250 mg/kg diet) to inhibit PIN was assessed in two independent sets of experiments. T + E induced multiple PIN in the dorsolateral prostate (DLP) of 80-100% of the animals. DHEA and DHEA 8354 did not affect the incidence but decreased the multiplicity of PIN in the DLP, from 3.2 +/- 1.0 in control group to 1.5 +/- 1.0 in the low-dose and to 1.6 +/- 0.6 in the high-dose group for DHEA (P<0.05 and P<0.02, respectively), and to 1.9 +/- 0.8 in the high-dose (P<0.05) DHEA 8354. Both agents did not affect PIN in anterior prostate, seminal vesicles or ventral prostate. In a second experiment, DFMO and oltipraz were found not effective in inhibiting PIN. In this study, we provide new evidence that PIN in Noble rats, induced by continuous T + E treatment, is a useful intermediate endpoint for determining the efficacy of DHEA and other potential chemopreventive agents. The hormonal pathogenesis, high multiplicity, short latency, preferential location in the DLP, similarity in morphology and biology to PIN of human prostate, and the sensitivity to agents that suppress prostate carcinogenesis, makes PIN in Noble rats a promising intermediate endpoint for chemoprevention studies.

Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography-electrospray mass spectrometry.

Employing high-performance liquid chromatography-electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC-MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, beta,beta-dimethyl-gamma-(hydroxymethyl)-gamma-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1+/-2.7 and 9.4+/-3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC50 values 0.24+/-0.02 and 2.16+/-0.31 microM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.