Madeleine Moussa

Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices

Two citrus flavonoids, hesperetin and naringenin, found in oranges and grapefruit, respectively, and four noncitrus flavonoids, baicalein, galangin, genistein, and quercetin, were tested singly and in one-to-one combinations for their effects on proliferation and growth of a human breast carcinoma cell line, MDA-MB-435. The concentration at which cell proliferation was inhibited by 50% (IC50), based on incorporation of [3H]thymidine, varied from 5.9 to 140 micrograms/ml for the single flavonoids, with the most potent being baicalein. IC50 values for the one-to-one combinations ranged from 4.7 micrograms/ml (quercetin + hesperetin, quercetin + naringenin) to 22.5 micrograms/ml (naringenin + hesperetin). All the flavonoids showed low cytotoxicity (> 500 micrograms/ml for 50% cell death). Naringenin is present in grapefruit mainly as its glycosylated form, naringin. These compounds, as well as grapefruit and orange juice concentrates, were tested for their ability to inhibit development of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Two experiments were conducted in which groups of 21 rats were fed a semipurified diet containing 5% corn oil and were given a 5-mg dose of DMBA intragastrically at approximately 50 days of age while in diestrus. One week later, individual groups were given double-strength grapefruit juice or orange juice or fed naringin or naringenin at levels comparable to that provided by the grapefruit juice; in the second experiment, the rats were fed a semipurified diet containing 20% corn oil at that time. As expected, rats fed the high-fat diet developed more tumors than rats fed the low-fat diet, but in both experiments tumor development was delayed in the groups given orange juice or fed the naringin-supplemented diet compared with the other three groups. Although tumor incidence and tumor burden (grams of tumor/rat) were somewhat variable in the different groups, rats given orange juice had a smaller tumor burden than controls, although they grew better than any of the other groups. These experiments provide evidence of anticancer properties of orange juice and indicate that citrus flavonoids are effective inhibitors of human breast cancer cell proliferation in vitro, especially when paired with quercetin, which is widely distributed in other foods.

A New Three-Dimensional Ultrasound Microimaging Technology for Preclinical Studies Using a Transgenic Prostate Cancer Mouse Model

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Prostate Histopathology: Learning Tissue Component Histograms for Cancer Detection and Classification

Radical prostatectomy is performed on approximately 40% of men with organ-confined prostate cancer. Pathologic information obtained from the prostatectomy specimen provides important prognostic information and guides recommendations for adjuvant treatment. The current pathology protocol in most centers involves primarily qualitative assessment. In this paper, we describe and evaluate our system for automatic prostate cancer detection and grading on hematoxylin & eosin-stained tissue images. Our approach is intended to address the dual challenges of large data size and the need for high-level tissue information about the locations and grades of tumors. Our system uses two stages of AdaBoost-based classification. The first provides high-level tissue component labeling of a superpixel image partitioning. The second uses the tissue component labeling to provide a classification of cancer versus noncancer, and low-grade versus high-grade cancer. We evaluated our system using 991 sub-images extracted from digital pathology images of 50 whole-mount tissue sections from 15 prostatectomy patients. We measured accuracies of 90% and 85% for the cancer versus noncancer and high-grade versus low-grade classification tasks, respectively. This system represents a first step toward automated cancer quantification on prostate digital histopathology imaging, which could pave the way for more accurately informed postprostatectomy patient care.

Registration of prostate histology images to ex vivo MR images via strand-shaped fiducials.

To present and evaluate a method for registration of whole‐mount prostate digital histology images to ex vivo magnetic resonance (MR) images.

Prostate: Registration of Digital Histopathologic Images to in Vivo MR Images Acquired by Using Endorectal Receive Coil

PURPOSE To develop and evaluate a technique for the registration of in vivo prostate magnetic resonance (MR) images to digital histopathologic images by using image-guided specimen slicing based on strand-shaped fiducial markers relating specimen imaging to histopathologic examination. MATERIALS AND METHODS The study was approved by the institutional review board (the University of Western Ontario Health Sciences Research Ethics Board, London, Ontario, Canada), and written informed consent was obtained from all patients. This work proposed and evaluated a technique utilizing developed fiducial markers and real-time three-dimensional visualization in support of image guidance for ex vivo prostate specimen slicing parallel to the MR imaging planes prior to digitization, simplifying the registration process. Means, standard deviations, root-mean-square errors, and 95% confidence intervals are reported for all evaluated measurements. RESULTS The slicing error was within the 2.2 mm thickness of the diagnostic-quality MR imaging sections, with a tissue block thickness standard deviation of 0.2 mm. Rigid registration provided negligible postregistration overlap of the smallest clinically important tumors (0.2 cm(3)) at histologic examination and MR imaging, whereas the tested nonrigid registration method yielded a mean target registration error of 1.1 mm and provided useful coregistration of such tumors. CONCLUSION This method for the registration of prostate digital histopathologic images to in vivo MR images acquired by using an endorectal receive coil was sufficiently accurate for coregistering the smallest clinically important lesions with 95% confidence.

Identification of trichloroethylene and its metabolites in human seminal fluid of workers exposed to trichloroethylene.

We have investigated the potential of the male reproductive tract to accumulate trichloroethylene (TCE) and its metabolites, including chloral, trichloroethanol (TCOH), trichloroacetic acid (TCA), and dichloroacetic acid (DCA). Human seminal fluid and urine samples from eight mechanics diagnosed with clinical infertility and exposed to TCE occupationally were analyzed. In in vivo experimental studies, TCE and its metabolites were determined in epididymis and testis of mice exposed to TCE (1000 ppm) by inhalation for 1 to 4 weeks. In other studies, incubations of monkey epididymal microsomes were performed in the presence of TCE and NADPH. Our results showed that seminal fluid from all eight subjects contained TCE, chloral, and TCOH. DCA was present in samples from two subjects, and only one contained TCA. TCA and/or TCOH were also identified in urine samples from only two subjects. TCE, chloral, and TCOH were detected in murine epididymis after inhalation exposure with TCE for 1 to 4 weeks. Levels of TCE and chloral were similar throughout the entire exposure period. TCOH levels were similar at 1 and 2 weeks but increased significantly after 4 weeks of TCE exposure. Chloral was identified in microsomal incubations with TCE in monkey epididymis. CYP2E1, a P450 that metabolizes TCE, was localized in human and monkey epididymal epithelium and testicular Leydig cells. These results indicated that TCE is metabolized in the reproductive tract of the mouse and monkey. Furthermore, TCE and its metabolites accumulated in seminal fluid, and suggested associations between production of TCE metabolites, reproductive toxicity, and impaired fertility.

Knockin of SV40 Tag oncogene in a mouse adenocarcinoma of the prostate model demonstrates advantageous features over the transgenic model

Prostate cancer (CaP) is the most common cancer in adult men in North America. Since there is no naturally occurring prostate cancer in the mouse, preclinical studies stipulate for the establishment of a genetically manipulated mouse CaP model with features close to the human situation. In view of the limitations of transgenic technique-derived CaP models, herein we report the first application of knockin technology to establish a new mouse adenocarcinoma prostate model (PSP-KIMAP) by targeting of SV40 Tag to a prostate tissue-specific gene, PSP94 (prostate secretory protein of 94 amino acids). In order to demonstrate its novelty, we compared KIMAP to a PSP94 gene-directed transgenic mouse adenocarcinoma of the prostate (PSP-TGMAP) model. The CaP development of the PSP-KIMAP mice started almost immediately after puberty at 10 weeks of age from mouse prostatic intraepithelial neoplasia (mPIN) with microinvasion to well-differentiated CaP, and demonstrated a close-to-human kinetics of prolonged tumor growth and a predominance of well and moderately differentiated tumors. The invasive nature of KIMAP model was demonstrated by multitissue metastases (lymph node, lung and liver etc) and also by immunohistochemical study of multiple invasive prostate tumor markers. PSP-KIMAP model is responsive to androgen deprivation (castration). The knockin technology in our KIMAP model demonstrates highly predictive CaP development procedures and many advantageous features, which the traditional transgenic technique-derived CaP models could not reach for both basic and clinical studies. These features include the high stability of both phenotype and genotype, highly synchronous prostate cancer development, high and precise prostate tissue targeting and with no founder line variation. The differences between the two CaP models were attributed to the introduction of a single endogenous knockin mutation, resulting in a CaP model self-regulated and controlled by a prostate gene promoter/enhancer of PSP94.

PSP94 EXPRESSION AFTER ANDROGEN DEPRIVATION THERAPY: A COMPARATIVE STUDY WITH PROSTATE SPECIFIC ANTIGEN IN BENIGN PROSTATE AND PROSTATE CANCER

PURPOSE To examine the clinical use of PSP94 (prostate secretory protein of 94 amino acids) as an androgen independent marker, we conducted a comparative study of prostate samples including benign tissue and cancers which did and did not have androgen deprivation. MATERIALS AND METHODS Among 163 radical prostatectomy cases 75 had androgen deprivation before operation, while surgery was performed in the remainder without prior hormone treatment. Considering the pathological up grading following hormone therapy, contiguous sections from radical prostatectomy samples were stained for PSP94 and prostate specific antigen (PSA) by immunohistochemistry, and equivalent tumor foci were evaluated by assessing the intensity and extent of the staining. RESULTS In untreated benign prostate tissue PSP94 and PSA staining was positive and identical in all sections in the no pretreatment group. However, PSP94 expression in the androgen deprivation group was significantly higher than PSA in intensity (p = 0.0005) and extent (p = 0.034). In untreated cancer cases PSP94 intensity and extent demonstrated strong inverse association with Gleason grade (p <0.0001). In contrast, PSA expression was high in every grade, resulting in no statistical association with tumor grade. In the androgen deprivation group PSA staining was decreased in every grade compared to the no pretreatment group. On the other hand, PSP94 expression was decreased in grade 3 tumor foci but increased in grades 4 and 5 tumor foci compared with samples of the corresponding grade in the no pretreatment group (p = 0.0034). CONCLUSIONS PSP94 expression in benign prostate persists under androgen deprivation compared to PSA. PSP94 synthesis in high grade tumor appears to be activated in the absence of androgen stimulation, indicating the possible alternative pathways in the regulation of PSP94.

Molecular targeted enhanced ultrasound imaging of flk1 reveals diagnosis and prognosis potential in a genetically engineered mouse prostate cancer model.

Molecular imaging techniques used to detect the initiation of disease have the potential to provide the best opportunity for early treatment and cure. This report aimed at testing the possibility that Flk1+ (vascular endothelial growth factor receptor 2), a crucial angiogenesis factor of most tumor cells, could be a molecular targeted imaging marker for the diagnosis and prognosis of cancer. We performed Flk1-targeted microbubble-enhanced ultrasound (US) imaging of prostate cancer in a genetically engineered mouse model with normal-appearing intact US (negative) prostates and with three different tumor sizes (small, medium, and large). Higher levels of Flk1+ molecular signals were identified in the intact US (negative) prostate group by US-targeted imaging and immunohistochemical analysis. The increase in Flk1+ expression occurred prior to the angiogenesis switch-on phase and vascularity peak. After this peak accumulation stage of Flk1+ molecules, lower and stabilized levels of Flk1+ signals were maintained together with tumor growth from small, to medium, to large size. In a longitudinal observation in a subset (n = 5) of mice with established tumors, elevated Flk1+ signals were observed in tissues surrounding the prostate cancer, for example, the ipsilateral boundary zones between two developing tumor lobes, new tumor blood vessel recruits, the urethra border, and the pelvic node basin. The potential of Flk1-targeted US imaging as a predictive imaging tool was confirmed by correlation studies of three-dimensional US B-mode imaging, gross pathology, and histology analyses. The results of the application in a genetically engineered mouse model with prostate cancer of molecular Flk1-targeted US imaging support the contention that Flk1 can be used as a molecular imaging marker for small tumors undetectable by microimaging and as a molecular diagnostic and prognosis marker for tumor metastasis and progression.

Establishment of a Serum Tumor Marker for Preclinical Trials of Mouse Prostate Cancer Models

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.