TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1.     

Cannabinoid receptor agonist-induced apoptosis of human prostate cancer cells LNCaP proceeds through sustained activation of ERK1/2 leading to G1 cell cycle arrest

We have recently shown that the expression levels of both cannabinoid receptors CB(1) and CB(2) are higher in human prostate cancer cells than in normal prostate epithelial cells, and treatment of LNCaP cells with WIN-55,212-2 (a mixed CB(1)/CB(2) agonist) resulted in inhibition of cell growth and induction of apoptosis (Sarfaraz, S., Afaq, F., Adhami, V. M., and Mukhtar, H. (2005) Cancer Res. 65, 1635-1641). This study was conducted to understand the mechanistic basis of these effects. Treatment of LNCaP cells with WIN-55,212-2 (1-10 microm; 24 h) resulted in: (i) an arrest of the cells in the G(0)/G(1) phase of the cell cycle; (ii) an induction of p53 and p27/KIP1; (iii) down-regulation of cyclins D1, D2, E; (iii) decrease in the expression of cdk-2, -4, and -6; (iv) decrease in protein expression of pRb; (v) down-regulation of E2F (1-4); and (vi) decrease in the protein expression of DP1 and DP2. Similar effects were also observed when androgen-independent PC3 cells were treated with WIN-55,212-2 (5-30 microm). We further observed sustained up-regulation of ERK1/2 and inhibition of PI3k/Akt pathways in WIN-55,212-2-treated cells. Inhibition of ERK1/2 abrogated WIN-55,212-2-indued cell death suggesting that sustained activation of ERK1/2 leads to cell cycle dysregulation and arrest of cells in G(0)/G(1) phase subsequently leading to an induction of apoptosis. Further, WIN-55,212-2 treatment of cells resulted in a dose-dependent increase in Bax/Bcl-2 ratio in such a way that favors apoptosis. The induction of apoptosis proceeded through down-regulation of caspases 3, 6, 7, and 9 and cleavage of poly (ADP-ribose) polymerases. Based on these data we suggest that cannabinoid receptor agonists should be considered as novel agents for the management of prostate cancer.     

Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.     

Down-regulation of phosphoglucomutase 3 mediates sulforaphane-induced cell death in LNCaP prostate cancer cells

Background

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables that exerts anti-oxidant, anti-inflammatory, anti-cancer and radio-sensitizing activities. Nonetheless, the mechanism responsible for SFN-induced cell death is not fully understood. In the present study, anti-cancer mechanism of SFN was elucidated in LNCaP prostate cancer cells.

Results

SFN exerted cytotoxicity and increased TUNEL positive cells in a concentration-dependent manner in LNCaP cells. Proteomics study revealed that levels of nine proteins including tubulin β-2, phosphoglucomutase-3 (PGM3), melanoma-derived leucine zipper containing extra-nuclear factor, activin A type I receptor precursor, smoothelin-A, KIA0073, hypothetical protein LOC57691 and two unnamed proteins were changed over 8 folds in SFN treated LNCaP cells compared to untreated control. We have further confirmed that SFN reduced PGM3 expression with western blotting and showed that PGM3 siRNA enhanced cytotoxicity demonstrated by cell morphology and TUNEL assays in LNCaP cells.

Conclusion

Taken together, these findings suggest that PGM3 plays a role in mediating SFN-induced cell death in LNCaP cells, and is a potential molecular therapeutic target for prostate cancer.     

Modulation of the retinoic acid-induced cell apoptosis and differentiation by the human TR4 orphan nuclear receptor

In our previous studies, the TR4 orphan nuclear receptor (TR4) has been demonstrated to suppress retinoic acid (RA)-induced transactivation via a negative feedback control mechanism and in situ analysis showed that TR4 is extensively expressed in mouse brain, especially in regions where the cells are proliferating. To further study the potential roles of TR4 during cell differentiation, a tetracycline-inducible system with anti-sense TR4 in teratocarcinoma P19 cell lines was generated to analyze the retinoic acid-induced differentiation of these cells. The results indicated that the expression of TR4 reduced by doxycycline anti-sense TR4 would alter the retinoic acid-induced differentiation pathway that results in the changes of cell morphology and cell cycle profile. Unexpectedly, our data further indicated that the RA-induced apoptosis, judging by DNA fragmentation, could also be altered by the induction of anti-sense TR4. Together, these findings provide the first in vivo evidence that an orphan nuclear receptor, such as TR4, may play major roles in the RA-mediated apoptosis or differentiation in P19 cells.     

Recent advances in the TR2 and TR4 orphan receptors of the nuclear receptor superfamily

The human testicular receptor 2 (TR2) and TR4 orphan receptors are two evolutionarily related proteins belonging to the nuclear receptor superfamily. Numerous TR2 and TR4 variants and homologs have been identified from different species, including vertebrates (e.g. human, murine, rabbit, fish, and amphibian) and invertebrates (e.g. Drosophila, sea urchin, and nematode) since TR2 was initially isolated over a decade ago. Specific tissue distribution, genomic organization, and chromosomal assignment of both orphan receptors have been investigated. In order to reveal the physiological functions played by both TR2 and TR4, upstream modulators of TR2 and TR4 gene expression, their downstream target gene regulation, feedback mechanisms, and differential modulation mediated by the recruitment of other nuclear receptors and coregulators have been investigated. Studies summarized in the present report have provided unexpected insights into the TR2 and TR4 functions in a variety of biological processes. The essential and difficult tasks of identifying orphan receptor ligands, agonist/antagonist assignment, their physiological functions, and mechanisms of action will continue to challenge nuclear receptor researchers in the future.     

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells

Background

The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCF^SKP2 ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer.

Results

We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCF^SKP2. Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block.

Conclusions

Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies.     

The nuclear orphan receptor TR4 promotes proliferation of myeloid progenitor cells.

Nuclear receptors represent key regulators in cell proliferation, differentiation, and development. Here we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues and have analyzed the impact of TR4 in this cell compartment. We show that TR4, when ectopically expressed in bone marrow cells via retrovirus vector, promotes proliferation of myeloid progenitor cells. Cells represent promyelocytes as judged by morphological features, expression of cell surface molecules, and specific markers like Mim-1 and CAAT/enhancer binding protein beta. We also demonstrate that the growth promoting activity of TR4 is not exclusively dependent on its association with DNA, because expression of a mutated TR4 version devoid of its DNA binding domain exhibits a similar proliferative potential as wild-type TR4. In conclusion, these data position the orphan receptor TR4 as an important regulator of myeloid progenitor cell proliferation and development.     

Physiologically attainable concentrations of lycopene induce mitochondrial apoptosis in LNCaP human prostate cancer cells

Prostate cancer is the second leading cause of cancer deaths among men in the United States. Studies show that people with diets rich in tomato-based foods have reduced risks of cancer, viz., prostate cancer. This is attributed, in part, to lycopene, the most abundant carotenoid in tomatoes. Thus, we studied the effect of lycopene at physiologically attainable concentrations on apoptosis, cellular proliferation, and necrosis in LNCaP human prostate cancer cells. Cells at 37 degrees C and >80% confluency were treated with media alone (0.32% tetrahydrofuran vehicle) or with increasing concentrations (0.3-3.0 microM) of lycopene overnight. After washing monolayers, analyses by high-performance liquid chromatography (HPLC) showed that cellular accumulation of lycopene was 5.5 +/- 0.8, 14.0 +/- 3.2, and 36.7 +/- 12.3 pmole/10(6) cells for 0.3, 1.0, and 3.0 muM, respectively, and not detected in control cells. Lycopene did not alter cellular proliferation because bromodeoxyuridine (BrdU) incorporation and cell numbers were identical among groups. However, results of a 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that mitochondrial function decreased 61%-83% with increasing concentrations of lycopene (P < 0.001). Cytotoxicity and necrosis did not contribute to this effect because lactate dehydrogenase (LDH) release (1.5%-1.8%) and trypan blue exclusion (89%-93%) were similar. Subsequently, we demonstrated that increasing concentrations of lycopene significantly (P < 0.05) reduced mitochondrial transmembrane potential, induced the release of mitochondrial cytochrome c, and increased annexin V binding, confirming induction of apoptosis. Thus, lycopene at physiologically relevant concentrations did not affect cellular proliferation or promote necrosis but clearly altered mitochondrial function and induced apoptosis in LNCaP human prostate cancer cells.     

Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells

Prostate cancer is the most commonly diagnosed neoplasm in men. LNCaP cells continue to possess many of the molecular characteristics of in situ prostate cancer. These cells lack ras mutations, and mitogen-activated protein kinase (MAPK) is not extensively phosphorylated in these cells. To determine the effects of ras/raf/MAPK pathway activation in these cells, we transfected LNCaP cells with an activatable form of c-raf-1(deltaRaf-1:ER). Activation of deltaRaf-1:ER, with resultant MAPK activation, reduced plating efficiency and soft agarose cloning efficiency 30-fold in LNCaP cells. Cell cycle distribution showed an accumulation of cells in G1 and was associated with the induction of CDK inhibitor p21WAF1/CIP1 at the protein and mRNA levels. p21WAF1/CIP1 mRNA stability was increased after deltaRaf-1:ER activation. In addition, activated deltaRaf-1:ER induced the senescence associated-beta-galactosidase in LNCaP cells. These data demonstrate that raf activation can activate growth inhibitory pathways leading to growth suppression in prostate carcinoma cells and also suggest that raf/MEK/MAPK pathway activation, rather than inhibition, may be a therapeutic target for some human prostate cancer cells.