D Cvetkovic

Increased hypoxia correlates with increased expression of the angiogenesis marker vascular endothelial growth factor in human prostate cancer.

OBJECTIVES To test the hypothesis that increasing levels of hypoxia are associated with increased expression of vascular endothelial growth factor (VEGF) in prostate cancer by correlating the level of median tissue oxygenation in human prostate tumors with the immunohistochemically determined level of VEGF expression. METHODS Custom-made Eppendorf oxygen microelectrodes were used to quantitate the pO(2) levels in prostate tumors of 13 men undergoing radical prostatectomy. All pO(2) measurements were performed under fluorine-based general anesthesia. Paraffin-embedded tumor tissue from these men was analyzed to measure the level of VEGF expression by immunohistochemical staining. The significance of the associations between the pO(2) levels and VEGF staining were determined by the Pearson correlations. RESULTS The range of the median pO(2) levels (based on between 97 and 129 individual measurements) among 13 prostate tumors was 0.5 to 44.9 mm Hg. The blinded comparison of pO(2) levels and VEGF staining intensity demonstrated a significant correlation between increasing hypoxia and the percentage of cells staining positive for VEGF (r = -0.721, P = 0.005). This correlation was also significant when pO(2) levels were compared with the overall immunoreactive score, which takes into account staining intensity (r = -0.642, P = 0.018). CONCLUSIONS To our knowledge, this is the first study demonstrating a significant association between increasing levels of hypoxia and increased expression of the angiogenesis marker VEGF in human prostate carcinoma. The results of our study further support the exploration of antiangiogenesis strategies for the treatment of human prostate cancer.

Early events in ovarian oncogenesis

Ovarian cancer represents the most lethal of the gynecological neoplasms. The molecular and genetic events associated with early ovarian oncogenesis are still largely unknown, thus contributing to the lack of reliable biomarkers for disease detection. Since the majority of ovarian tumors are diagnosed at an advanced stage, the availability of early ovarian cancer tissue samples for molecular analyses is very limited. In this review, problems encountered in the study of early ovarian cancer are presented, along with the controversies concerning precursor lesions and stepwise progression towards ovarian malignancy. Experimental modeling in the development of ovarian cancer is also described, as well as genetic and epigenetic alterations associated with early ovarian cancer. Lastly, examples of technological advances in the study of early ovarian cancer are discussed. Hopefully, the increasing knowledge about molecular and genetic events involved in the early stages of ovarian tumorigenesis will provide the basis for management of ovarian cancer in the future.

Decreased expression of retinol-binding proteins is associated with malignant transformation of the ovarian surface epithelium.

We have developed a modified form of suppression subtractive hybridization (SSH) that allows multiple specimens of distinct phenotypic groups to be compared for consistent differences in gene expression. We applied this system to identify genes that were expressed in normal rat ovarian surface epithelial (ROSE) cells but whose expression was lost/downregulated in four independently transformed rat ovarian cancer cell lines. Northern blot analysis using 14 of 28 nonredundant cDNA fragments from this difference library showed that the mRNA transcripts were present in normal ROSE cells but lost or markedly downregulated in four related transformed cell lines. Of particular interest, cellular retinol-binding protein 1 (CRBP1) and retinol-binding protein (RBP), two genes whose products are involved in retinol transport and metabolism, were found to be downregulated in this ovarian cancer model system. To determine if this change had relevance to human ovarian cancer, we evaluated a series of human ovarian cancer cell lines and a limited number of frozen human ovarian tumors and found lost or decreased expression of CRBP1 and RBP relative to expression in human ovarian surface epithelial (HOSE) cells. We hypothesize that the loss of CRBP1 and RBP expression disrupts retinol metabolism and retinoic acid production, which may facilitate the occurrence of genetic damage leading to the malignant transformation of the ovarian surface epithelium, the cells from which ovarian cancer arises.

Vitamin A Metabolism is Impaired in Human Ovarian Cancer

OBJECTIVES We have previously reported that loss in expression of a protein considered critical for vitamin A homeostasis, cellular retinol-binding protein 1 (CRBP1), is an early event in ovarian carcinogenesis. The aim of the present study was to determine if loss of vitamin A metabolism also occurs early in ovarian oncogenesis. METHODS We assessed CRBP1 expression by immunohistochemistry in ovaries prophylactically removed from women with a genetic risk for ovarian cancer. Furthermore, we investigated the ability of normal, immortalized but nontumorigenic, and tumorigenic human ovarian epithelial cells to synthesize retinoic acid and retinaldehyde when challenged with a physiological dose of retinol, and determined expression levels of the retinoid-related genes, RARalpha, RXRalpha, CRABP1, CRABP2, RALDH1 and RALDH2 in these cells. RESULTS Immunohistochemistry revealed loss of CRBP1 expression in potentially preneoplastic lesions in prophylactic oophorectomies. HPLC analysis of vitamin A metabolism showed production of retinoic acid in four independent, normal human ovarian surface epithelial (HOSE) cell cultures upon exposure to retinol. However, only one of two SV40-immortalized HOSE cell lines made RA, while none of the ovarian carcinoma cell lines produced detectable RA due to complete loss of RALDH2. CONCLUSIONS The impaired conversion of retinol to RA in ovarian cancer cells and decreased CRBP1 protein expression in prophylactic oophorectomies support our hypothesis that concomitant losses of vitamin A metabolism and CRBP1 expression contribute to ovarian oncogenesis.

Effects on gene expression in rat liver after administration of RXR agonists: UAB30, 4-methyl-UAB30, and Targretin (Bexarotene).

Examination of three retinoid X receptor (RXR) agonists [Targretin (TRG), UAB30, and 4-methyl-UAB30 (4-Me-UAB30)] showed that all inhibited mammary cancer in rodents and two (TRG and 4-Me-UAB30) strikingly increased serum triglyceride levels. Agents were administered in diets to female Sprague-Dawley rats. Liver RNA was isolated and microarrayed on the Affymetrix GeneChip Rat Exon 1.0 ST array. Statistical tests identified genes that exhibited differential expression and fell into groups, or modules, with differential expression among agonists. Genes in specific modules were changed by one, two, or all three agonists. An interactome analysis assessed the effects on genes that heterodimerize with known nuclear receptors. For proliferator-activated receptor α/RXR-activated genes, the strongest response was TRG > 4-Me-UAB30 > UAB30. Many liver X receptor/RXR-related genes (e.g., Scd-1 and Srebf1, which are associated with increased triglycerides) were highly expressed in TRG and 4-Me-UAB30- but not UAB30-treated livers. Minimal expression changes were associated with retinoic acid receptor or vitamin D receptor heterodimers by any of the agonists. UAB30 unexpectedly and uniquely activated genes associated with the aryl hydrocarbon hydroxylase (Ah) receptor (Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1). Based on the Ah receptor activation, UAB30 was tested for its ability to prevent dimethylbenzanthracene (DMBA)-induced mammary cancers, presumably by inhibiting DMBA activation, and was highly effective. Gene expression changes were determined by reverse transcriptase-polymerase chain reaction in rat livers treated with Targretin for 2.3, 7, and 21 days. These showed similar gene expression changes at all three time points, arguing some steady-state effect. Different patterns of gene expression among the agonists provided insight into molecular differences and allowed one to predict certain physiologic consequences of agonist treatment.

MR-guided pulsed high intensity focused ultrasound enhancement of docetaxel combined with radiotherapy for prostate cancer treatment

The purpose of this study is to evaluate the efficacy of the enhancement of docetaxel by pulsed focused ultrasound (pFUS) in combination with radiotherapy (RT) for treatment of prostate cancer in vivo. LNCaP cells were grown in the prostates of male nude mice. When the tumors reached a designated volume by MRI, tumor bearing mice were randomly divided into seven groups (n = 5): (1) pFUS alone; (2) RT alone; (3) docetaxel alone; (4) docetaxel + pFUS; (5) docetaxel + RT; (6) docetaxel + pFUS + RT, and (7) control. MR-guided pFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5T GE MR scanner. Animals were treated once with pFUS, docetaxel, RT or their combinations. Docetaxel was given by i.v. injection at 5 mg kg(-1) before pFUS. RT was given 2 Gy after pFUS. Animals were euthanized 4 weeks after treatment. Tumor volumes were measured on MRI at 1 and 4 weeks post-treatment. Results showed that triple combination therapies of docetaxel, pFUS and RT provided the most significant tumor growth inhibition among all groups, which may have potential for the treatment of prostate cancer due to an improved therapeutic ratio.

Quantitative study of focused ultrasound enhanced doxorubicin delivery to prostate tumor in vivo with MRI guidance

PURPOSE The purpose of this study was to investigate the potential of MR-guided pulsed focused ultrasound (pFUS) for the enhancement of drug uptake in prostate tumors in vivo using doxorubicin (Dox). METHODS An antitumor drug Dox, an orthotopic animal prostate tumor model using human prostate cancer, LNCaP cell line, and a clinical FUS treatment system (InSightec ExAblate 2000) with a 1.5T GE MR scanner were used in this study. First, experiments on a tissue mimic phantom to determine the optimal acoustic power and exposure durations with a 10% duty cycle and a 1 Hz pulse rate were performed. The temperature variation was monitored using real-time MR thermometry. Second, tumor-bearing animals were treated with pFUS. There were three groups (n = 8/group): group 1 received pFUS + Dox (10 mg/kg i.v. injection immediately after pFUS exposure), group 2 received Dox only (10 mg/kg i.v. injection), and group 3 was a control. Animals were euthanized 2 h after the pFUS treatment. The Dox concentration in the treated tumors was measured by quantifying fluorescent tracers using a fluorometer. Third, the histological changes of tumors with and without pFUS treatments were evaluated. Finally, experiments were performed to study the spatial drug distribution in tumors after the pFUS treatment, in which two animals received pFUS + Dox, two animals received Dox only, and one animal was used as control. Two hours following the treatment, animals were euthanized and processed. The Dox distribution was determined using a fluorescence microscope. RESULTS Parametric measurements using a tissue phantom showed that the temperature increased with an increasing acoustic power (from 10 to 50 W) or sonication duration (from 10 to 60 s) with a given acoustic frequency of 1 MHz, duty cycle 10%, and pulse rate 1 Hz. A set of ultrasound parameters was identified with which the temperature elevation was less than 5 °C, which was used for nonthermal pFUS sonication. Increased Dox concentration (14.9 ± 2.5 μg/g) was measured in the pFUS-treated group compared to the Dox-only group (9.5 ± 1.6 μg/g), indicating an approximate 60% increase with p = 0.05. The results were consistent with the increased spatial drug distributions by fluorescence imaging. Histological analysis showed increased extravasation in pFUS-treated prostate tumors suggesting increased drug delivery with pFUS. CONCLUSIONS The results showed that pFUS-enhanced drug uptake in prostate tumors was significant. This increased uptake may be due to increased extravasation by pFUS. Optimal pFUS parameters may exist to maximize the drug uptake, and this study using Dox demonstrated a quantitative method for such systematic parametric studies. In addition, this study may provide useful data for the potential application of pFUS-mediated Dox delivery for prostate tumor therapy.

Local Tumor Control and Normal Tissue Toxicity of Pulsed Low-Dose Rate Radiotherapy for Recurrent Lung Cancer An In Vivo Animal Study

Objectives: This study investigates (1) local tumor control and (2) normal tissue toxicity of pulsed low-dose rate radiotherapy (PLDR) for recurrent lung cancer. Methods: For study 1, nude mice were implanted with A549 tumors and divided into the following 3 groups: (1) control (n = 10), (2) conventional radiotherapy (RT; n = 10), and (3) PLDR (n = 10). Tumor-bearing mice received 2 Gy daily dose for 2 consecutive days. Weekly magnetic resonance imaging was used for tumor growth monitoring. For study 2, 20 mice received 8 Gy total body irradiation either continuously (n = 10) or 40 × 0.2 Gy pulses with 3-minute intervals (n = 10). Results: For study 1, both conventional RT and PLDR significantly inhibited the growth of A549 xenografts compared with the control group (>35% difference in the mean tumor volume; P < .05). The PLDR results were slightly better than conventional RT (8% difference in the mean tumor volume; P > .05). For study 2, the average weight was 20.94 ± 1.68 g and 25.69 ± 1.27 g and the survival time was 8 days and 12 days for mice treated with conventional RT and PLDR (P < .05), respectively. Conclusion: This study showed that PLDR could control A549 tumors as effectively as conventional RT, and PLDR induced much less normal tissue toxicity than conventional RT. Thus, PLDR would be a good modality for recurrent lung cancers. Advances in Knowledge: This article reports our results of an in vivo animal investigation of PLDR for the treatment of recurrent cancers, which may not be eligible for treatment because of the dose limitations on nearby healthy organs that have been irradiated in previous treatments. This was the first in vivo study to quantify the tumor control and normal tissue toxicities of PLDR using mice with implanted tumors, and our findings provided evidence to support the clinical trials that employ PLDR treatment techniques.

Tissue TGF-β expression following conventional radiotherapy and pulsed low-dose-rate radiation

ABSTRACT The release of inflammatory cytokines has been implicated in the toxicity of conventional radiotherapy (CRT). Transforming growth factor β (TGF-β) has been suggested to be a risk marker for pulmonary toxicity following radiotherapy. Pulsed low-dose rate radiotherapy (PLDR) is a technique that involves spreading out a conventional radiotherapy dose into short pulses of dose with breaks in between to reduce toxicities. We hypothesized that the more tolerable toxicity profile of PLDR compared with CRT may be related to differential expression of inflammatory cytokines such as TGF-β in normal tissues. To address this, we analyzed tissues from mice that had been subjected to lethal doses of CRT and PLDR by histology and immunohistochemistry (IHC). Equivalent physical doses of CRT triggered more cellular atrophy in the bone marrow, intestine, and pancreas when compared with PLDR as indicated by hematoxylin and eosin staining. IHC data indicates that TGF-β expression is increased in the bone marrow, intestine, and lungs of mice subjected to CRT as compared with tissues from mice subjected to PLDR. Our in vivo data suggest that differential expression of inflammatory cytokines such as TGF-β may play a role in the more favorable normal tissue late response following treatment with PLDR.

TH-C-217BCD-01: Best in Physics (Imaging) - Evaluation of Apoptosis and Proliferation in Non-Thermal Pulsed HIFU Treated Mouse Prostate Tumors

Purpose: The underlying mechanism of non‐thermal effects of pulsed high intensity focused ultrasound (pFUS) on normal and tumor tissues is not well understood. Our recent studies showed significant prostate cancercell killing in vitro and significant prostate tumorgrowth delay in vivo after pFUS treatment. We hypothesized that these effects of non‐thermal pFUS are due to an increase in cell apoptosis and decrease in cell proliferation. Therefore, the purpose of this study was to analyze the biomarkers of apoptosis and proliferation in prostate tumors after pFUS exposures. Methods: An orthotopic prostate tumor model was established in nude mice. Prostate tumors were sonicated with MR guided pFUS (1MHz, 5W acoustic power, 5Hz frequency; 0.1duty cycle) for 60 sec for each sonication with temperature <42°C (InSightec ExAblate 2000 with a 1.5T GE MR scanner). Untreated tumors were used as controls. The mice were sacrificed at predetermined times up to 7 days following therapy. Tumors were processed for light microscopic examination with H&E staining and immunohistochemical staining for caspase 3 (a marker of apoptosis) and Ki67 (aproliferation marker) expression. Results: Light microscopy revealed the absence of thermal damage and acute destruction of tumor tissues exposed to pFUS. The microvessel walls in the tissues remained intact. There was no change in the extent of hemorrhage upon pFUS treatment over time. The apoptotic index, defined as a percentage of apoptotic cells per total number of cells, peaked at 24 hours after FUS treatment relative to control. There was no dramatic difference in the proliferation indices between different time points. Conclusions: Our results suggested that non‐thermal pulsed pFUS induced caspase 3‐mediated apoptosis and did not produce any thermal damage in the prostate tumor tissues. We are performing additional studies to evaluate blood vessel density and cellular DNA damage upon pFUS treatment.