Genetic polymorphisms in oestrogen receptor-binding sites affect clinical outcomes in patients with prostate cancer receiving androgen-deprivation therapy.

BACKGROUND: Accumulating evidence indicates that oestrogens have significant direct effects on normal prostate development and carcinogenesis. The majority of the biological activities of oestrogens are mediated through the oestrogen receptor (ER), which functions as a hormone-inducible transcription factor to regulate target gene expression by binding to oestrogen response elements (EREs) in the regulatory regions of target genes. Sequence variants in EREs might affect the ER-ERE interaction and subsequent physiological activities. Therefore, we tested whether common single-nucleotide polymorphisms (SNPs) inside EREs are related to the clinical outcomes of androgen-deprivation therapy (ADT) in men with prostate cancer. METHODS: We systematically evaluated 49 ERE SNPs predicted using a genome-wide database in a cohort of 601 men with advanced prostate cancer treated with ADT. The prognostic significance of these SNPs on disease progression, prostate cancer-specific mortality (PCSM) and all-cause mortality (ACM) after ADT was assessed using Kaplan-Meier analysis and a Cox regression model. RESULTS: Based on multiple hypothesis testing, BNC2 rs16934641 was found to be associated with disease progression; in addition, TACC2 rs3763763 was associated with PCSM, and ALPK1 rs2051778 and TACC2 rs3763763 were associated with ACM. These SNPs remained significant in multivariate analyses that included known clinicopathological predictors. Moreover, a combined genotype effect on ACM was observed when ALPK1 rs2051778 and TACC2 rs3763763 were analysed in combination. Patients with a greater number of unfavourable genotypes had a shorter time to ACM during ADT (P for trend <0.001). CONCLUSION: The incorporation of ERE SNPs into models with known predictors might improve outcome prediction in patients with prostate cancer receiving ADT.

Antizyme, a natural ornithine decarboxylase inhibitor, induces apoptosis of haematopoietic cells through mitochondrial membrane depolarization and caspases' cascade.

Antizymes delicately regulate ornithine decarboxylase (ODC) enzyme activity and polyamine transportation. One member of the family, antizyme-1, plays vital roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. However, the question of how does it participate in the cell apoptotic mechanism is still unsolved. To elucidate the contribution of human antizyme-1 in haematopoietic cell death, we examine whether inducible overexpression of antizyme enhances apoptotic cell death. Antizyme reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells, acute T leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G(1) appearance, loss of mitochondrial membrane potential (Deltapsi( m )), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following conditional antizyme overexpression, all protein levels of cyclin-dependent kinases (Cdks) and cyclins are not significantly reduced, except cyclin D, before their entrance into apoptotic cell death. However, introduced cyclin D1 into Jurkat T tetracycline (Tet)-On cell system still couldn't rescue cells from apoptosis. Antizyme doesn't influence the expression of tumor suppressor p53 and its downstream p21, but it interferes in the expressions of Bcl-2 family. Inducible antizyme largely enters mitochondria resulting in cytochrome c release from mitochondria to cytosol following Bcl-xL decrease and Bax increase. According to these data, we suggest that antizyme induces apoptosis mainly through mitochondria-mediated and cell cycle-independent pathway. Furthermore, antizyme induces apoptosis not only by Bax accumulation reducing the function of the Bcl-2 family, destroying the Deltapsi( m ), and releasing cytochrome c to cytoplasm but also by the activation of apoptosomal caspase cascade.

Increasing ornithine decarboxylase activity is another way of prolactin preventing methotrexate-induced apoptosis: crosstalk between ODC and BCL-2.

Prolactin has more than 300 separate functions including affecting mammary growth, differentiation, secretion and anti-apoptosis. In the previous studies, prolactin induced Bcl-2 expression to prevent apoptosis and also provoked the activity of ornithine decarboxylase (ODC). Our previous data showed that ODC overexpression upregulates Bcl-2 and prevents tumor necrosis factor alpha (TNF-alpha)- and methotrexate (MTX)-induced apoptosis. Here, we further investigate whether prolactin prevents MTX-induced apoptosis through inducing ODC activity and the relationship between ODC and Bcl-2 upon prolactin stimulation. Prolactin prevented MTX-induced apoptosis in a dose-dependent manner in HL-60 cells. Following prolactin stimulation, ODC enzyme activity also shows an increase in a dose-dependent manner, expressing its maximum level at 3 h, and rapidly declining thereafter. Prolactin-induced ODC activity is completely blocked by a protein kinase C delta (PKCdelta) inhibitor, rottlerin. However, there are no changes in the expressions of ODC mRNA and protein level after prolactin stimulus. It indicates that prolactin may induce ODC activity through the PCKdelta pathway. Besides, Bcl-2 expresses within 1 h of prolactin treatment and this initiating effect of prolactin is not inhibited by alpha-difluoromethylornithine (DFMO). However, Bcl-2 is further enhanced following prolactin stimulation for 4 h and this enhancement is blocked by DFMO. Bcl-2 has no effect on ODC activity and protein levels, but ODC upregulates Bcl-2, which is inhibited by DFMO. Overall, there are two different forms of prolactin effect, it induces Bcl-2 primarily, and following this it stimulates ODC activity. Consequently induced ODC activity further enhances the expression of Bcl-2. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, ODC activity is one basis for the anti-apoptotic mechanisms of prolactin. A Bcl-2 inhibitor, HA14-1, together with DFMO, completely blocks the anti-apoptotic effects of prolactin. These results suggest that increasing ODC activity is another way of prolactin preventing MTX-induced apoptosis and that this induction of ODC activity enhances the expression of Bcl-2 strongly enough to bring about the anti-apoptotic function.

Cross-resistance and combined cytotoxic effects of paclitaxel and cisplatin in bladder cancer cells.

PURPOSE: We studied the cross-resistance and combined cytotoxic effects of cisplatin and paclitaxel in bladder cancer cells in vitro. MATERIALS AND METHODS: The cytotoxicity of the 2 agents alone or in combination were studied in the bladder cancer cell line NTUB1 and the 2 sublines NTUB1/P, which is cisplatin resistant, and NTUB1/T, which is paclitaxel resistant, using the microculture tetrazolium assay. Schedule dependence of the 2-drug combination was assayed using 3 treatment schedules, including 1 concurrent and 2 sequential exposures. RESULTS: The mean cisplatin concentration plus or minus standard error of the means inhibiting 50% of the growth of NTUB1, NTUB1/P and NTUB1/T was 1.9 +/- 0.19, 19.3 +/- 2.33 and 2.1 +/- 0.15 microM., respectively, and the mean paclitaxel concentration inhibiting 50% of the growth of the 3 cell lines was 30 +/- 3.9, 1,033 +/- 120 and 110 +/- 15 nM., respectively. NTUB1/P had strong cross-resistance to paclitaxel. In contrast, NTUB1/T was as sensitive as NTUB1 to cisplatin. On median effect analysis the combined effects of the 2 agents given concurrently were sub-additive in the low fraction affected range of 0.1 to 0.3 and additive in the median to high fraction affected range of 0.4 to 1.0 in the 3 cell lines. Combined cytotoxicity was more synergistic when paclitaxel was given 24 hours earlier than cisplatin. The effects were less synergistic when cisplatin was given before paclitaxel. This phenomenon was noted in sensitive and resistant cells. CONCLUSIONS: In our bladder cancer cell model cisplatin resistant cells have strong cross-resistance to paclitaxel, whereas paclitaxel resistant cells are sensitive to cisplatin. The combined effects may be optimized by sequential use of the 2 agents, preferably paclitaxel given 24 hours before cisplatin. Our results have clinical implications for the treatment of bladder cancer.

Characterization of chemoresistance mechanisms in a series of cisplatin-resistant transitional carcinoma cell lines.

We explored the mechanisms of cisplatin resistance in a series of bladder transitional carcinoma cells that are either sensitive or progressively resistant to cisplatin. Resistant lines were raised by chronic exposure of the parental cells to progressively increased concentrations of cisplatin. The cisplatin IC50s of the sensitive and the three resistant cells were 4.3, 25.0, 40.4, and 52.2 microM, respectively. The expressions of glutathione S-transferase pi (GST-pi) and multidrug resistance-associated protein (MRP) were enhanced in a dose-response manner as cells acquired progressive cisplatin resistance. Expression of mdr-1 transcript was detected in the three resistant lines but not in the sensitive line. Glutathione contents were increased in resistant cells, yet the trend of increase did not reach statistical significance (p = 0.061). In conclusion, transitional carcinoma cells may gain cisplatin resistance through multiple pathways including up-regulation of GST-pi, MRP and possibly mdr-1. Glutathione contents may play a less significant role in cisplatin chemoresistance.

Suppression of transcription factor NF-kappaB activity by Bcl-2 protein in NIH3T3 cells: implication of a novel NF-kappaB p50-Bcl-2 complex for the anti-apoptotic function of Bcl-2.

Bcl-2 can suppress apoptosis by controlling genes that encode proteins required for programmed cell death and by interference with peroxidative damage. Overexpression of Bcl-2 in NIH3T3 cells can prevent GSNO-induced (S-nitrosoglutathione-induced) apoptosis. The experimental results indicated that activation of NF-kappaB by GSNO is involved in inducing apoptosis. Surprisingly, we found that Bcl-2 delayed the release of IkB by formation of a Bcl-2-NF-kappaB complex (p50-p65-IkappaB) in the cytoplasm during cell apoptosis. Furthermore, a novel Bcl-2-p50 complex was found in the nucleus. These features were only observed in Bcl-2-transfected cells but not in the parental NIH3T3 cells. Overexpression of Bcl-2 suppressed the levels of c-myc, a target gene of NF-kappaB, and influenced the DNA-binding activity of NF-kappaB during GSNOinduced apoptosis. We suggest that the Bcl-2-p50 complex inhibits NF-kappaB DNA-binding activity by competing with the p65-p50 heterodimer for the DNA-binding site in the nucleus. Finally, it has been demonstrated that the anti-apoptotic potential of Bcl-2 may be attributed to its complexing with p50 in the nucleus that leads to blockage of nuclear gene expression.

Suppression of N-methyl-N'-nitro-N-nitrosoguanidine- and S-nitrosoglutathione-induced apoptosis by Bcl-2 through inhibiting glutathione-S-transferase pi in NIH3T3 cells.

In this study, both NIH3T3 and Bcl-2 transfected NIH3T3 cells were examined for their propensity to undergo nitroso compound-induced apoptosis. Bcl-2-expressing NIH3T3 prevented N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)- and S-nitrosoglutathione (GSNO)-induced apoptosis as compared with the control NIH3T3 cells. Flow cytometry revealed that NIH3T3 cells treated with MNNG undergo apoptotic death, which occurred after G2-M arrest in the second cycle of cell proliferation. The mechanism of MNNG-induced NIH3T3 cells apoptosis was observed throughout the activation of caspase-3 protease, PARP degradation and cytochrome c release; it was independent of p53 activation. Glutathione-S-transferanse pi (GST pi) is activated through the transcription activation of antioxidant response element (ARE) during MNNG- and GSNO-induced cell apoptosis. Moreover, overexpression of Bcl-2 in NIH3T3 cells can prevent these features of cell death. Furthermore, both MNNG- and GSNO-induced apoptosis of NIH3T3 cells were accompanied with a decrease in the level of glutathione (GSH); whereas Bcl-2 overexpression led to an increase in total cellular glutathione. MNNG was metabolized rapidly to nitric oxide that reacted with glutathione under the catalysis of GSH transferase in NIH3T3 cell to form GSNO. In short, the production of GSNO in cells was found capable of apoptosis initiation while the overexpression of Bcl-2 can prevent MNNG-mediated cell apoptosis through the elevation of glutathione levels.

Inhibition of eleven mutagens by various tea extracts, (-)epigallocatechin-3-gallate, gallic acid and caffeine.

The antimutagenic properties of various tea extracts (green tea, pauchong tea, oolong tea and black tea) and their components including (-)-epigallocatechin-3-gallate (EGCG), gallic acid and caffeine were examined by the Ames test. The antimutagenic activity of the green tea extract against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), folpet and monocrotophos was greater than those of pouchong, oolong and black tea extracts. The antimutagenic effects of tea extracts against 2-acetylaminofluorene (AAF) decreased as follows: oolong tea > pauchong tea > black tea > green tea. Furthermore, black tea showed a greater antimutagenic activity against benzo[a]pyrene (BP). The pauchong tea showed a stronger inhibitory effect against 9-aminoacridine (9AA) and aflatoxin B1 (AFB1) than other tea extracts. EGCG markedly suppressed the direct-acting mutagenicity of MNNG, N-nitroso-N-methylurea (MNU), captan, and folpet which were alkylating agents and fungicides. Similarly, gallic acid, the major component of black tea strongly inhibited the mutagenicity of 9AA, and moderately inhibited the mutagenicity of MNNG and folpet. The caffeine was less active. EGCG and gallic acid perhaps could act as nucleophiles to scavenge the electrophilic mutagens. Taken together, these results suggest that formation of different metabolites during various stages of tea fermentation may affect antimutagenic potencies against different types of chemical mutagens.

Comparative investigation on the mutagenicities of organophosphate, phthalimide, pyrethroid and carbamate insecticides by the Ames and lactam tests.

The Salmonella lactam test is a newly developed method for detecting genotoxins. This technique is based on the ability of DNA damaging agents to reverse expression of the beta-lactamase gene, an important gene that enables microbes to resist beta-lactam antibiotics. A construct p-SELECT Control DNA plasmid containing a beta-lactamase gene site was constructed in many mutant forms, including point and frameshift mutants. These mutant constructs were introduced into Salmonella tester strains whose mutagenicity is based on their ability to reverse expression of the beta-lactamase gene. Fourteen pesticides were evaluated for genotoxicity using our newly developed Salmonella typhimurium strains JK947 and JK3, which are useful for detecting base substitution mutations. Six pesticides, namely allethrin, captan, folpet, monocrotophos, acephate and carbofuran, proved highly mutagenic in strain JK947, while the first four pesticides were more weakly mutagenic in strain JK3. In comparison, results from the Ames test show strain JK947 to be more sensitive to these pesticides than strains TA100 and TA1535. Strains TA98 and JK1 proved insensitive to allethrin, captan, folpet, acephate, carbofuran and monocrotophos. Among the many advantages of the lactam test are: large numbers of cells can treated and the test is operationally simple and inexpensive; revertant colonies form faster in the lactam test (16 h) than in the Ames test (48 h); the lactam test can detect mutagens present in biological specimens contaminated by histidine and biotin, samples that may give false positive results in the Ames test.

A new mutagenicity assay method for frameshift mutagens based on deleting or inserting a guanosine nucleotide in the beta-lactamase gene.

The conventional method of site-directed mutagenesis was used to develop two Salmonella strains, JK-1 and JK-2, for detecting frameshift mutagens. The JK-1 strain was derived from Salmonella typhimurium TA1537 strain transformed by a mutant construct. A guanosine nucleotide was inserted between nucleotide residues 312 and 313 of the beta-lactamase gene. The JK-2 strain was obtained by the same procedure, but a guanosine nucleotide in position 315 of the beta-lactamase gene was deleted. The strains were tested with ten frameshift mutagens and the revertants were selected by ampicillin resistance. Representative mutagens including 2-nitrofluorene (2-NF), 2-acetylaminofluorene (AAF), 9-aminoacridine (9-AA), 2,7-diaminofluorene (2,7-DAF) and 2-methoxy-6-chloro-9-(3-(ethyl-2-chloro-ethyl)-aminopropylamino)acridine (ICR-170) were more potent in the JK-1 strain than the JK-2 strain, and the number of revertant colonies were dose related. Under the same conditions, the ampicillin test was more sensitive than the Ames test. Other types of compounds such as 2-methoxy-6-chloro-9-(2-chloroethylaminopropylamino)acridine (ICR-191), benzo[a]pyrene (BP), 4-nitroquinoline N-oxide (4-NQNO), hycanthone and aflatoxin B1 (AFB1) were not as mutagenic to these new strains. The method is quite promising for studying certain specific frameshift mutagens, but more chemical mutagens should be tested to validate its applicability and reproducibility in general use.