Methylation of SRD5A2 promoter predicts a better outcome for castration-resistant prostate cancer patients undergoing androgen deprivation therapy

PURPOSE: To determine whether SRD5A2 promoter methylation is associated with cancer progression during androgen deprivation therapy (ADT) in CRPC. PATIENTS AND METHODS: In a Local CRPC cohort, 42 prostatic specimens were collected from patients who were diagnosed as CRPC and underwent transurethral resection of the prostate (TURP) at Massachusetts General Hospital (MGH). In a metastatic CRPC (Met CRPC) cohort, 12 metastatic biopsies were collected from CRPC patients who would be treated with abiraterone plus dutasteride (Clinical Trial NCT01393730). As controls, 36 benign prostatic specimens were collected from patients undergoing prostate reduction surgery for symptoms of bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH). The methylation status of cytosine-phosphate-guanine (CpG) site(s) at SRD5A2 promoter regions was tested. RESULTS: Compared with benign prostatic tissue, CRPC samples demonstrated higher SRD5A2 methylation in the whole promoter region (Local CRPC cohort: P \u3c 0.001; Met CRPC cohort: P \u3c 0.05). In Local CRPC cohort, a higher ratio of methylation was correlated with better OS (R2 = 0.33, P = 0.013). Hypermethylation of specific regions (nucleotides -434 to -4 [CpG# -39 to CpG# -2]) was associated with a better OS (11.3+/-5.8 vs 6.4+/-4.4 years, P = 0.001) and PFS (8.4+/-5.4 vs 4.5+/-3.9 years, P = 0.003) with cutoff value of 37.9%. Multivariate analysis showed that SRD5A2 methylation was associated with OS independently (whole promoter region: P = 0.035; specific region: P = 0.02). CONCLUSION: Our study demonstrate that SRD5A2 methylation in promoter regions, specifically at CpG# -39 to -2, is significantly associated with better survival for CRPC patients treated with ADT. Recognition of epigenetic modifications of SRD5A2 may affect the choices and sequence of available therapies for management of CRPC

Inhibition of TNF-α Improves the Bladder Dysfunction That Is Associated With Type 2 Diabetes

Diabetic bladder dysfunction (DBD) is common and affects 80% of diabetic patients. However, the molecular mechanisms underlying DBD remain elusive because of a lack of appropriate animal models. We demonstrate DBD in a mouse model that harbors hepatic-specific insulin receptor substrate 1 and 2 deletions (double knockout [DKO]), which develops type 2 diabetes. Bladders of DKO animals exhibited detrusor overactivity at an early stage: increased frequency of nonvoiding contractions during bladder filling, decreased voided volume, and dispersed urine spot patterns. In contrast, older animals with diabetes exhibited detrusor hypoactivity, findings consistent with clinical features of diabetes in humans. The tumor necrosis factor (TNF) superfamily genes were upregulated in DKO bladders. In particular, TNF-α was upregulated in serum and in bladder smooth muscle tissue. TNF-α augmented the contraction of primary cultured bladder smooth muscle cells through upregulating Rho kinase activity and phosphorylating myosin light chain. Systemic treatment of DKO animals with soluble TNF receptor 1 (TNFRI) prevented upregulation of Rho A signaling and reversed the bladder dysfunction, without affecting hyperglycemia. TNFRI combined with the antidiabetic agent, metformin, improved DBD beyond that achieved with metformin alone, suggesting that therapies targeting TNF-α may have utility in reversing the secondary urologic complications of type 2 diabetes

Development of nonviral gene delivery and of anti-gene reagents

Although significant progress has been made in the basic science and applications of various non-viral gene delivery systems, the majority of non-viral approaches are still much less efficient than viral vectors, especially for in vivo gene delivery, Therefore, non-viral gene delivery must be developed in order to increase the efficiency of in vivo delivery. Moreover, a novel anti-gene reagent was developed for sequence-specific gene silencing. Peptide Nucleic Acid (PNA), a mimic of oligonucleotide with a neutral pseudopeptide backbone consisting of repeating N- (2-amino-ethyl)-glycine units, can invade double-stranded DNA to generate a stable single-strand D-loop. Locked Nucleic Acid (LNA) are synthetic analogs of nucleic acids, which contain a bridging methylene carbon between the 2 and 4 positions of the ribose ring. Over the years, a variety of chemically modified small molecules have been developed. Among them, PNA and LNA have shown great promise for a number of applications. In order to further develop a technology for linking functional entities to non-viral vectors, the Bioplex technology, we investigated how hybridization of PNA to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to DNA strands. Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNA, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found enhanced kinetics when PNA binding to overlapping sites on opposite DNA strands were added, without the use of further chemically modified bases in the PNAs (I). We also generated a novel, sequence-specific anti-gene molecule Zorro LNA , in which a 14-mer LNA binds to the coding strand, while a 16-mer connected LNA binds to the template strand. Our data suggest that the Zorro LNA induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in mammalian cells. This offers a novel type of anti-gene drug, which is easy to synthesize (II). We also found that the Zorro LNA construct efficiently inhibited pol III-dependent transcription in a cellular context, including in vivo in a mouse model. Thus, this new form of gene silencer could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs (III). Finally, In order to further evaluate the in vivo activity of Zorro LNA and trace its long-term effect in inhibiting gene expression, we inserted two adjacent Zorro LNA-binding sites flanked by loxP sites within the transcribed region of a reporter gene. As previously observed, after Zorro LNA was hybridized to the plasmid, it significantly induced gene silencing in the mammalian cells. Both in transfected cells and in mouse model the silencing effect was lost when Cre-recombinase expressing plasmids were subsequently transfected. This suggests that binding of Zorro LNA is stable over a period of days

Optimization of Ultrasound-Assisted Extraction of Polyphenols from Ilex latifolia Using Response Surface Methodology and Evaluation of Their Antioxidant Activity

The polyphenolic extract of Ilex latifolia (PEIL) exhibits a variety of biological activities. An evaluation of the parameters influencing the ultrasonic extraction process and the assessment of PEIL antioxidant activity are presented herein. Response surface methodology (RSM) was used to optimize the experimental conditions for the polyphenols ultrasonic-assisted extraction (UAE) from the leaves of Ilex latifolia. We identified the following optimal conditions of PEIL: ethanol concentration of 53%, extraction temperature of 60 °C, extraction time of 26 min and liquid–solid ratio of 60 mL/g. Using these parameters, the UAE had a yield of 35.77 ± 0.26 mg GAE/g, similar to the value we predicted using RSM (35.864 mg GAE/g). The antioxidant activity of PEIL was assessed in vitro, using various assays, as well as in vivo. We tested the effects of various doses of PEIL on D-galactose induced aging. Vitamin C (Vc) was used as positive control. After 21 days of administration, we measured superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, malondialdehyde (MDA) levels in mouse serum and liver tissue. The results demonstrated that the PEIL exhibits potent radical scavenging activity against 1,1-diphenyl-2-picrythydrazyl (DPPH∙), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+), and hydroxyl (∙OH) radicals. The serum concentrations of SOD and GSH-Px were higher, and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging group (p < 0.01), and the activity of MDA was lower than those of the model group (p < 0.01). The liver concentrations of SOD and GSH-Px were higher (p < 0.05), and MDA levels were lower, in the medium- and high-dose PEIL-treated groups than those in the aging control group (p < 0.01). These results suggest that optimizing the conditions of UAE using RSM could significantly increase the yield of PEIL extraction. PEIL possesses strong antioxidant activity and use as a medicine or functional food could be further investigated

Metformin inhibits the proliferation of benign prostatic epithelial cells

Objective: Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods: BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results: Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the expression of IGF-1R in epithelial cells. Metformin abrogated the proliferation of benign prostatic epithelial cells promoted by 3T3 conditioned medium. Conclusions: Our study demonstrates that metformin inhibits the proliferation of benign prostatic epithelial cells by suppressing the expression of IGF-1R and IGF-1 secretion in stromal cells. Metformin lowers the G2/M cell population and simultaneously increases the G0/G1 population. Findings here might have significant clinical implications in management of BPH patients treated with metformin

Metformin represses cancer cells via alternate pathways in N-cadherin expressing vs. N-cadherin deficient cells

Metformin has emerged as a potential anticancer agent. Here, we demonstrate that metformin plays an anti-tumor role via repressing N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Ectopic-expression of N-cadherin develops metformin-resistant cancer cells, while suppression of N-cadherin sensitizes cancer to metformin. Manipulation of AMPK expression does not alter sensitivity of cancer to metformin. We show that NF-kappaB is a downstream molecule of N-cadherin and metformin regulates NF-kappaB signaling via suppressing N-cadherin. Moreover, we also suggest that TWIST1 is an upstream molecule of N-cadherin/NF-kappaB signaling and manipulation of TWIST1 expression changes the sensitivity of cancer cells to metformin. In contrast to the cells that express N-cadherin, in N-cadherin deficient cells, metformin plays an anti-tumor role via activation of AMPK. Ectopic expression of N-cadherin makes cancer more resistant to metformin. Therefore, we suggest that metformin's anti-cancer therapeutic effect is mediated through different molecular mechanism in wild-type vs. deficient N-cadherin cancer cells. At last, we selected 49 out of 984 patients’ samples with prostatic cancer after radical prostatectomy (selection criteria: Gleason score ≥ 7 and all patients taking metformin) and showed levels of N-cadherin, p65 and AMPK could predict post-surgical recurrence in prostate cancer after treatment of metformin

Underlying data for Fig 3A, 3D, 3E, 3F and 3G.

Correction: Metformin inhibits the proliferation of benign prostatic epithelial cells</p

Metformin regulates cell cycle and the expression of proteins regulating cell cycle.

<p>A: Representative immunoblot analysis of cyclin D1, D2 and D3 in BPH-1 cells 48 hours after treatment of indicated dose of metformin. GAPDH was used as a loading control. B: The effect of 48-hour treatment of 100 ng/mL IGF-1 and 5 mM metformin on the expression of cyclin D1, D2 and D3 in BPH-1 cells. C-D: Flow cytometry analysis of proliferating BPH-1 (C) and P69 (D) cells 48 hours after the addition of 100 ng/mL IGF-1, 5 mM metformin or both. E-F: The population of G0/G1 cells in BPH-1 (E) and P69 (F) cells (n = 3). *, <i>P</i><0.05 compared with the control group; #, <i>P</i><0.05 compared with the IGF-1 treated group. CTR: non-treated control, Met: metformin.</p

Underlying data for Fig 4A and 4B.

Correction: Metformin inhibits the proliferation of benign prostatic epithelial cells</p

Quantification data for Fig 4C and 4D.

Correction: Metformin inhibits the proliferation of benign prostatic epithelial cells</p