Vivian X. Fu

Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphatidylinositol 3′-kinase/Akt pathway and Bcl-2 family proteins

Prostate cancer is a major health problem in the U.S. and the available treatment and surgical options have proven to be inadequate in controlling the mortality and morbidity associated with this disease. It is therefore necessary to intensify our efforts to better understand this disease and develop novel approaches for its prevention and treatment. This study was conducted to evaluate the chemopreventive/antiproliferative potential of resveratrol (trans-3,4′,5,-trihydroxystilbene) against prostate cancer and its mechanism of action. Treatment with resveratrol (0–50 μmol/L for 24 hours) resulted in a significant (a) decrease in cell viability, (b) decrease of clonogenic cell survival, (c) inhibition of androgen (R1881)-stimulated growth, and (d) induction of apoptosis in androgen-responsive human prostate carcinoma (LNCaP) cells. Interestingly, at similar concentrations, resveratrol treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cells. Furthermore, our data showed that resveratrol-treatment resulted in significant dose-dependent inhibition in the constitutive expression of phosphatidylinositol 3′-kinase and phosphorylated (active) Akt in LNCaP cells. Resveratrol treatment for LNCaP cells was also found to result in a significant (a) loss of mitochondrial membrane potential, (b) inhibition in the protein level of antiapoptotic Bcl-2, and (c) increase in proapoptotic members of the Bcl-2 family, i.e., Bax, Bak, Bid, and Bad. Taken together, our data suggested that resveratrol causes an inhibition of phosphatidylinositol 3′-kinase/Akt activation that, in turn, results in modulations in Bcl-2 family proteins in such a way that the apoptosis of LNCaP cells is promoted. Based on these studies, we suggest that resveratrol could be developed as an agent for the management of prostate cancer. [Mol Cancer Ther 2006;5(5):1335–41]

Novel Pathways Associated with Bypassing Cellular Senescence in Human Prostate Epithelial Cells

Cellular senescence forms a barrier that inhibits the acquisition of an immortal phenotype, a critical feature in tumorigenesis. The inactivation of multiple pathways that positively regulate senescence are required for immortalization. To identify these pathways in an unbiased manner, we performed DNA microarray analyses to assess the expression of 20,000 genes in human prostate epithelial cells (HPECs) passaged to senescence. These gene expression patterns were then compared with those of HPECs immortalized with the humanPapillomavirus 16 E7 oncoprotein. Senescent cells display gene expression patterns that reflect their nonproliferative, differentiated phenotype and express secretory proteases and extracellular matrix components. A comparison of genes transcriptionally up-regulated in senescence to those in which expression is significantly down-regulated in immortalized HPECs identified three genes: the chemokine BRAK,DOC1, and a member of the insulin-like growth factor axis,IGFBP-3. Expression of these genes is found to be uniformly lost in human prostate cancer cell lines and xenografts, and previously, their inactivation was documented in tumor samples. Thus, these genes may function in novel pathways that regulate senescence and are inactivated during immortalization. These changes may be critical not only in allowing cells to bypass senescence in vitrobut in the progression of prostate cancer in vivo.

Role of cyclin-dependent kinase inhibitors in the growth arrest at senescence in human prostate epithelial and uroepithelial cells

Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30–40 population doublings cells became growth-arrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point defined as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescence-associated β-galactosidase (SA-β-gal). Levels of p16INK4a and p57KIP2 rise in HUCs during progressive passages, whereas only p16 increases in HPEC cultures. The induced expression of p57, similar to p16, produces a senescent-like phenotype. pRB, cyclin D, p19INK4d and p27KIP1 decrease in both cell types. We find that p53, p21CIP1 and p15INK4b are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest, then return to low proliferating levels at terminal senescence. Analysis of p53, p21CIP1, p15INK4b, p16INK4a, and p57KIP2 reveals altered expression in immortalized, non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells. These results indicate: (i) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence, (ii) a distinct class of genes involved in the maintenance of senescence, and (iii) the frequent inactivation of these pathways during immortalization.

A methyl-deficient diet modifies histone methylation and alters Igf2 and H19 repression in the prostate.

Folate and methyl‐group deficiency has been linked to prostate cancer susceptibility, yet the mechanisms underlying these observations are incompletely understood. The region of the genome containing the imprinted genes insulin‐like growth factor 2 (Igf2) and H19, both of which display oncogenic functions, may be particularly sensitive to environmental influences.

Aging and Cancer-Related Loss of Insulin-like Growth Factor 2 Imprinting in the Mouse and Human Prostate

Loss of imprinting (LOI) is an epigenetic alteration involving loss of parental origin-specific expression at normally imprinted genes. A LOI for Igf2, a paracrine growth factor, is important in cancer progression. Epigenetic modifications may be altered by environmental factors. However, is not known whether changes in imprinting occur with aging in prostate and other tissues susceptible to cancer development. We found a LOI for Igf2 occurs specifically in the mouse prostate associated with increased Igf2 expression during aging. In older animals, expression of the chromatin insulator protein CTCF and its binding to the Igf2-H19 imprint control region was reduced. Forced down-regulation of CTCF leads to Igf2 LOI. We further show that Igf2 LOI occurs with aging in histologically normal human prostate tissues and that this epigenetic alteration was more extensive in men with associated cancer. This finding may contribute to a postulated field of cancer susceptibility that occurs with aging. Moreover, Igf2 LOI may serve as a marker for the presence of prostate cancer.

Alterations in the p16/pRb cell cycle checkpoint occur commonly in primary and metastatic human prostate cancer

We examined the status of a cell cycle checkpoint by immunohistochemically staining for p16 and pRb using multiple tissue arrays generated from 49 primary and 23 hormone-sensitive metastatic human prostate cancers. We find that p16, a cell cycle inhibitor, is paradoxically overexpressed in 83% of proliferating primary prostate cancers and increased expression correlates with a more rapid treatment failure (P=0.01) and a higher histologic grade (P=0.001). pRb staining is heterogeneous, loses expression infrequently (19%), and does not correlate with p16 expression. Loss of either p16 or pRb expression is found significantly (P=0.01) more commonly (55%) in metastatic specimens. The remarkable frequency of p16/pRb alterations and strong clinical associations implicates inactivation of this pathway as a critical determinant in prostate cancer progression.

Pennington Scientific Symposium on Mechanisms and Retardation of Aging

The Pennington Scientific Symposium on Mechanismsand Retardation of Aging was held in Baton Rouge,Louisiana, USA from May 4–6, 2003. The aim of thissymposium was to gather leaders in the field of aging todiscuss the latest theories and mechanisms of aging aswell as the life prolonging effects of calorie restriction(CR). The overall program and list of speakers ispresented in Appendix A. To encourage lively discussion,speakers were invited to bring one guest who had eithersimilar or opposing views to their own. During the firstsession, several views of aging with respect to theendocrine system, brain function, cardiovascular function,immune system and susceptibility to cancer werepresented. The second session focused on mechanismsof life extension by CR induced alterations in neuro-endocrine pathways, gene expression, mitochondrialenergy metabolism and oxidative stress. The programended with a lengthy round-table discussion to facilitatecommunication on how these studies could translate intoCR research in humans. During the round-table discus-sions, speakers and guests identified a few noteworthyhighlights of the presentations to be further emphasizedin the present report. More specifically, this report willfocus on three major hypotheses of aging presentedduring Session 1; hormonal regulation, DNA methylationand nutrient sensing. From Session 2, we report onpotential biomarkers of aging and the latest results fromthe ongoing CR trials in non-human primates.Session 1: Theories of Aging in Tissues and OrgansAging may be defined as the gradual and progressivedeterioration in function of an organism. However, it isunclear exactly why organisms age. Steven Austad beganthe symposium with an insightful lecture about the manyexisting theories of aging. He focused on the major causaltheories including: (1) for the good of the species, (2)somatic inevitability and (3) evolutionary aging. He alsopresented several mechanistic theories of aging; includingthose implicating oxidative stress, telomeres and endocrinedysfunction, and hypothesized that both causal and