Session III: What Have We Learned From Studies in Model Systems? Moderator: Dr. Susan Percival, University of Florida- Gainseville What Has Microarray Analysis Revealed About the Mechanisms of Action of Lycopene in Prostate Tumors? Dr. Karin Wertz, DSM Nutritional Products High tomato intake and plasma lycopene levels are associated with a reduced prostate cancer risk. The objective of this research project was to investigate two main questions: 1) Is lycopene, the main carotenoid in tomato, responsible for the observed effect of tomato consumption? 2) If so, by what mechanisms does lycopene contribute to the reduced risk of prostate cancer? The effects of lycopene on tumorous and normal rat prostate tissue were compared. The data suggest that in tumorous rat prostate tissue lycopene: 1) significantly increased the necrosis rate compared to a placebo group (37% versus 23%, respectively); 2) reduced androgen signaling; 3) decreased IGF-I expression; and 4) down-regulated interleukin-6 expression. In normal rat prostate tissue, lycopene: 1) had no effect on prostate growth; 2) reduced androgen signaling; 3) decreased IGF-I expression; and 4) down-regulated inflammatory signals. In summary, lycopene reduced androgen signaling in both normal and tumorous rat prostate tissue. Although the same metabolic pathway was affected, different enzymes were regulated at the transcriptional level, accompanied by down-regulation of the same steroid target genes. Both in normal and tumor tissue, lycopene decreased IGF-I expression and down-regulated inflammatory signals (with a stronger antiinflammatory effect in normal prostate tissue than in tumors). Dr. Wertz also discussed some preliminary unpublished data that suggest that lycopene has no systemic influence on androgen signaling and that the local antiandrogen effect is specific to the prostate. Discussion A participant asked if there were other genes that were also changed in response to lycopene. Dr. Wertz commented that she had presented the most consistent results. Within one pathway, there are often many opposing directions of gene regulation that can be difficult to understand. Dr. Wertz’s approach was to group the genes by pathways and metabolic functions, try to identify target genes on those pathways, and determine if the regulation “fits together.” A participant asked if there were measures of tumor growth besides necrosis and why such a rapidly growing cell line was used. He also asked about the effects of castration or androgen deprivation on the cell line. Dr. Wertz responded that she did not conduct any tumor histology and that the cell line was recommended by a collaborator who had worked with the model previously. She stated that the MatLyLu officially is an androgen-independent cell line, based on analysis from 20 years ago. It is difficult to explain the down-regulation of androgen target genes, however, if the tumor truly is androgen independent. The participant stated that the tumor is classically known to be androgen independent, but this finding could be verified by assessing the growth of the tumor in a castrated animal. The tumor is poorly differentiated histologically, which is consistent with an androgen-independent cell line. He questioned whether a subtle change of 20, 30, or 40 % in the expression of an androgen-metabolizing enzyme could bring about a demonstrable change in the growth of such a tumor. Dr. Wertz clarified that the tumor size was not changed. The participant replied that the more rapidly a transplantable tumor grows, the more necrosis is present. When the rapidly growing tumors are transplanted, they quickly outgrow their vascular supply and the central cells undergo necrosis. In the absence of histology, questions about the mechanism remain. Dr. Wertz stated that this research resulted in descriptive data—a readout of what is happening. The data should be fit together to build a working hypothesis. These experiments are not the end, but the start of testing that hypothesis. A comment was made that even androgen-independent cell lines are known to respond to androgen receptor-signaling. The participant asked if the entire tumor was homogenized for RNA. If the more necrotic tumors from the lycopene group were treated the same as the less necrotic tumors, there would be a shift in the cells being arrayed. Dying cells might have different gene expression because they are undergoing necrosis. Dr. Wertz responded that she cut out a slice. Such drastic gene regulations between treatment groups are not related to a few percent more or less necrotic cells. In addition, the same signaling pathways were affected in healthy tissue. Another participant stated that a change in tumor size might be observed with serial magnetic resonance imaging (MRI) and asked if repeat MRIs were done. Dr. Wertz replied that they did only one MRI. Insulin-like growth factor binding protein (IGFBP)-3 is known to be a major regulator of IGF-I function. A participant asked if Dr. Wertz checked for IGFBP-3 expression. Dr. Wertz replied that in this study, after lycopene supplementation, IGFBP-3 expression was not found. It may be more important to identify pathways that consistently are regulated in the same direction, rather than trying to identify single genes. The data support that something happens to the IGF-I axis. Dr. Wertz commented that there were 8,800 genes on the chips. Nutritional compounds often have effects between 20 and 50 percent up-regulation or down-regulation. Thresholds of different stringency should be used, depending in part on the number of chips per group (the more chips, the lower the threshold). The goal is consistency, rather than reading at a sharp threshold. The chip community is moving toward statistical modeling of all the data. Another participant stated that current research has shown that there is significant diurnal variation in nuclear transcription factors. He asked if it is valid to investigate a single time point, given the temporal response in nuclear transcription factors, or if multiple time points should be used. Dr. Wertz replied that multiple time points would be better, but it costs more to investigate temporal response. 2/05 NIH Meeting February 2005 NIH February 2005 NIH February 2005 NIH February 2005 NIH Remember we are NOT Doctors and have NO medical training. 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