A Peptide mimicking a region in proliferating cell nuclear antigen specific to key protein interactions is cytotoxic to breast cancer.

Proliferating cell nuclear antigen (PCNA) is a highly conserved protein necessary for proper component loading during the DNA replication and repair process. Proteins make a connection within the interdomain connector loop of PCNA, and much of the regulation is a result of the inherent competition for this docking site. If this target region of PCNA is modified, the DNA replication and repair process in cancer cells is potentially altered. Exploitation of this cancer-associated region has implications for targeted breast cancer therapy. In the present communication, we characterize a novel peptide (caPeptide) that has been synthesized to mimic the sequence identified as critical to the cancer-associated isoform of PCNA. This peptide is delivered into cells using a nine-arginine linking mechanism, and the resulting peptide (R9-cc-caPeptide) exhibits cytotoxicity in a triple-negative breast cancer cell line, MDA-MB-436, while having less of an effect on the normal counterparts (MCF10A and primary breast epithelial cells). The novel peptide was then evaluated for cytotoxicity using various in vivo techniques, including ATP activity assays, flow cytometry, and clonogenetic assays. This cytotoxicity has been observed in other breast cancer cell lines (MCF7 and HCC1937) and other forms of cancer (pancreatic and lymphoma). R9-cc-caPeptide has also been shown to block the association of PCNA with chromatin. Alanine scanning of the peptide sequence, combined with preliminary in silico modeling, gives insight to the disruptive ability and the molecular mechanism of action of the therapeutic peptide in vivo.

Small-molecule targeting of proliferating cell nuclear antigen chromatin association inhibits tumor cell growth.

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.

Hemostatic gelatin sponge is a superior matrix to matrigel for establishment of LNCaP human prostate cancer in nude mice.

BACKGROUND: Matrigels, solubilized basement membrane preparations, are often used to support tumor development in animal models. However, tumors formed by a mixture of tumor cells and Matrigel may vary significantly. The purpose of this study was to compare tumor development and growth of LNCaP human prostate cancer cells mixed with Matrigel or in gelatin sponges. METHODS: LNCaP cells were mixed with Matrigel or absorbed into VETSPON, a gelatin sponge, and inoculated into the subcutis of nude mice. Tumor incidence and growth rate were determined. Gene expression and cell growth and survival in tumor lesions were evaluated by immunohistochemistry (IHC), immunoblotting, and RT-PCR. RESULTS: All mice (12/12) inoculated with LNCaP cells in VETSPON produced tumors, compared to 70% (19/27) of mice injected with the cells with Matrigel. Tumor volume also varied less with VETSPON implants. No significant differences were observed in gene expression, cell growth, apoptosis, and microvessel density in tumors established from the two types of implants. However, in samples collected on days 1 and 4, more cells in Matrigel implants than those in VETSPON implants were stained positive for cleaved-caspase 3 and -PARP1. Expression of VEGF-A, HIF-1α, and Bcl-2 was elevated in the early VETSPON implants. CONCLUSION: These data indicate that VETSPON promotes tumor cell survival at the early stage of implantation and suggest that the gelatin sponge is superior to Matrigel in supporting development and progression of human prostate cancer in nude mice. This model should be useful for preclinical studies in nude mice using LNCaP cells.

Secretory phospholipase A2-IIa is a target gene of the HER/HER2-elicited pathway and a potential plasma biomarker for poor prognosis of prostate cancer.

BACKGROUND: Our previous study showed that prostate cancer cells overexpress and secrete secretory phospholipases A2 group IIa (sPLA2-IIa) and plasma sPLA2-IIa was elevated in prostate cancer patients. The current study further explored the underlying mechanism of sPLA2-IIa overexpression and the potential role of sPLA2-IIa as a prostate cancer biomarker. METHODS: Plasma and tissue specimens from prostate cancer patients were analyzed for sPLA2-IIa levels. Regulation of sPLA2-IIa expression by Heregulin-α was determined by Western blot and reporter assay. RESULTS: We found that Heregulin-α enhanced expression of the sPLA2-IIa gene via the HER2/HER3-elicited pathway. The EGFR/HER2 dual inhibitor Lapatinib and the NF-kB inhibitor Bortezomib inhibited sPLA2-IIa expression induced by Heregulin-α. Heregulin-α upregulated expression of the sPLA2-IIa gene at the transcriptional level. We further confirmed that plasma sPLA2-IIa secreted by mouse bearing human prostate cancer xenografts reached detectable plasma concentrations. A receiver operating characteristic (ROC) analysis of patient plasma specimens revealed that high levels of plasma sPLA2-IIa, with the optimum cutoff value of 2.0 ng/ml, were significantly associated with high Gleason score (8-10) relative to intermediate Gleason score (6-7) prostate cancers and advanced relative to indolent cancers. The area under the ROC curve (area under curve, AUC) was 0.73 and 0.74, respectively. CONCLUSION: We found that Heregulin-α, in addition to EGF, contributes to sPLA2-IIa overexpression in prostate cancer cells. Our findings support the notion that high levels of plasma sPLA2-IIa may serve as a poor prognostic biomarker capable of distinguishing aggressive from indolent prostate cancers, which may improve decision-making and optimize patient management.

Association of the CYP1B1 Leu432Val polymorphism with the risk of prostate cancer: a meta-analysis.

Cytochrome P450 1B1 (CYP1B1) is a key P450 enzyme involved in the metabolism of exogenous and endogenous substrates in endocrine-mediated tumors such as prostate cancer. The potential significance of nonsynonymous SNP Leu432Val (rs1056836) as a risk factor in prostate cancer has been extensively studied. The objective of this meta-analysis was to quantitatively summarize the association between CYP1B1 Leu432Val polymorphism and prostate cancer. All eligible studies were searched and acquired from the PubMed and ISI databases. Statistical analysis was performed by using the software STATA 11.0. Ten case-controlled studies from nine eligible publications were identified, which includes 6,668 subjects with 3,221 cases and 3,447 controls. Overall, no significant association was found between the CYP1B1 Leu432Val polymorphism and prostate cancer susceptibility for Val/Val vs Leu/Leu (OR = 1.07; 95% CI: 0.79-1.44; P = 0.67), Leu/Val vs Leu/Leu (OR = 1.05; 95% CI: 0.94-1.17; P = 0.42), Leu/Val + Val/Val vs Leu/Leu (OR = 1.07; 95% CI: 0.91-1.26; P = 0.40) and Val/Val vs Leu/Val + Leu/Leu (OR = 1.11; 95% CI: 0.86-1.44; P = 0.43). However, a higher risk was found among Asians in all genetic models (Val/Val vs Leu/Leu :OR = 2.48, 95% CI: 1.14-5.39, P = 0.02; Leu/Val vs Leu/Leu: OR = 1.40, 95% CI: 1.03-1.89, P = 0.03; Leu/Val + Val/Val vs Leu/Leu: OR = 1.51, 95% CI = 1.14-2.01, P = 0.004; Val/Val vs Leu/Val + Leu/Leu: OR = 2.50, 95% CI = 1.35-4.56, P = 0.004). We were not able to detect any association in the subgroup analysis by source of controls and genotyping method in all genetic models. In conclusion, this meta-analysis provides evidence that CYP1B1 Leu432Val polymorphism is not associated with prostate cancer risk overall with the exception in Asians.

Antiandrogen Therapy Radiosensitizes Androgen Receptor-Positive Cancers to 18F-FDG.

A subset (35%) of triple-negative breast cancers (TNBCs) expresses androgen receptor (AR) activity. However, clinical trials with antiandrogen drugs have shown limited efficacy, with about a 19% clinical benefit rate. We investigated the therapeutic enhancement of antiandrogens as radiosensitizers in combination with 18F-FDG in TNBC. Methods: We screened 5 candidate drugs to evaluate shared toxicity when combined with either 18F-FDG, x-rays, or ultraviolet radiation, at doses below their respective half-maximal inhibitory concentrations. Cytotoxic enhancement of antiandrogen in combination with 18F-FDG was evaluated using cell proliferation and DNA damage assays. Finally, the therapeutic efficacy of the combination treatment was evaluated in mouse tumor models of TNBC and prostate cancer. Results: Bicalutamide, an antiandrogen drug, was found to share similar toxicity in combination with either 18F-FDG or x-rays, indicating its sensitivity as a radiosensitizer to 18F-FDG. Cell proliferation assays demonstrated selective toxicity of combination bicalutamide-18F-FDG in AR-positive 22RV1 and MDA-MB-231 cells in comparison to AR-negative PC3 cells. Quantitative DNA damage and cell cycle arrest assays further confirmed radiation-induced damage to cells, suggesting the role of bicalutamide as a radiosensitizer to 18F-FDG-mediated radiation damage. Animal studies in MDA-MB-231, 22RV1, and PC3 mouse tumor models demonstrated significant attenuation of tumor growth through combination of bicalutamide and 18F-FDG in the AR-positive model in comparison to the AR-negative model. Histopathologic examination corroborated the in vitro and in vivo data and confirmed the absence of off-target toxicity to vital organs. Conclusion: These data provide evidence that 18F-FDG in conjunction with antiandrogens serving as radiosensitizers has utility as a radiotherapeutic agent in the ablation of AR-positive cancers.

Plasma secretory phospholipase A2-IIa as a potential biomarker for lung cancer in patients with solitary pulmonary nodules.

BACKGROUND: Five-year survival for lung cancer has remained at 16% over last several decades largely due to the fact that over 50% of patients are diagnosed with locally-advanced or metastatic disease. Diagnosis at an earlier and potentially curable stage is crucial. Solitary pulmonary nodules (SPNs) are common, but the difficulty lies in the determination of which SPN is malignant. Currently, there is no convenient and reliable biomarker effective for early diagnosis. Secretory phospholipase A2-IIa (sPLA2-IIa) is secreted into the circulation by cancer cells and may allow for an early detection of lung cancer. METHODS: Plasma samples from healthy donors, patients with only benign SPN, and patients with lung cancer were analyzed. Expression of sPLA2-IIa protein in lung cancer tissues was also determined. RESULTS: We found that the levels of plasma sPLA2-IIa were significantly elevated in lung cancer patients. The receiver operating characteristic curve analysis, comparing lung cancer patients to patients with benign nodules, revealed an optimum cutoff value for plasma sPLA2-IIa of 2.4 ng/ml to predict an early stage cancer with 48% sensitivity and 86% specificity and up to 67% sensitivity for T2 stage lung cancer. Combined sPLA2-IIa, CEA, and Cyfra21.1 tests increased the sensitivity for lung cancer prediction. High level of plasma sPLA2-IIa was associated with a decreased overall cancer survival. sPLA2-IIa was overexpressed in almost all non-small cell lung cancer and in the majority of small cell lung cancer by immunohistochemistry analysis. CONCLUSION: Our finding strongly suggests that plasma sPLA2-IIa is a potential lung biomarker to distinguish benign nodules from lung cancer and to aid lung cancer diagnosis in patients with SPNs.

Proliferating cell nuclear antigen directly interacts with androgen receptor and enhances androgen receptor­mediated signaling.

Androgen receptor (AR) and/or its constitutively active splicing variants (AR­Vs), such as AR­V7 and ARv567es, is required for prostate cancer cell growth and survival, and cancer progression. Proliferating cell nuclear antigen (PCNA) is preferentially overexpressed in all cancers and executes its functions through interaction with numerous partner proteins. The aim of the present study was to investigate the potential role of PCNA in the regulation of AR activity. An identical consensus sequence of the PCNA­interacting protein­box (PIP­box) was identified at the N­terminus of human, mouse and rat AR proteins. It was found that PCNA complexes with the full­length AR (AR­FL) and AR­V7, which can be attenuated by the small molecule PIP­box inhibitor, T2AA. PCNA also complexes with ARv567es and recombinant AR protein. The PCNA inhibitors, PCNA­I1S and T2AA, inhibited AR transcriptional activity and the expression of AR target genes in LNCaP­AI and 22Rv1 cells, but not in AR­negative PC­3 cells. The knockdown of PCNA expression reduced dihydrotestosterone­stimulated AR transcriptional activity and abolished the inhibitory effect of PCNA­I1S on AR activity. The PCNA inhibitor, PCNA­I1, exerted additive growth inhibitory effects with androgen deprivation and enzalutamide in cells expressing AR­FL or AR­FL/AR­V7, but not in AR­negative PC­3 cells. Finally, R9­AR­PIP, a small peptide mimicking AR PIP­box, was found to bind to GFP­PCNA at Kd of 2.73 µM and inhibit the expression of AR target genes, AR transcriptional activity and the growth of AR­expressing cells. On the whole, these data strongly suggest that AR is a PCNA partner protein and interacts with PCNA via the PIP­box and that targeting the PCNA­AR interaction may represent an innovative and selective therapeutic strategy against prostate cancer, particularly castration­resistant prostate cancers overexpressing constitutively active AR­Vs.

Secretory phospholipase A2-IIa is involved in prostate cancer progression and may potentially serve as a biomarker for prostate cancer.

The majority of prostate cancers are indolent, whereas a significant portion of patients will require systemic treatment during the course of their disease. To date, only high Gleason scores are best associated with a poor prognosis in prostate cancer. No validated serum biomarker has been identified with prognostic power. Previous studies showed that secretory phospholipase A2-IIa (sPLA2-IIa) is overexpressed in almost all human prostate cancer specimens and its elevated levels are correlated with high tumor grade. Here, we found that sPLA2-IIa is overexpressed in androgen-independent prostate cancer LNCaP-AI cells relative to their androgen-dependent LNCaP cell counterparts. LNCaP-AI cells also secrete significantly higher levels of sPLA2-IIa. Blocking sPLA2-IIa function compromises androgen-independent cell growth. Inhibition of the ligand-induced signaling output of the HER network, by blocking PI3K-Akt signaling and the nuclear factor-kappaB (NF-κB)-mediated pathway, compromises both sPLA2-IIa protein expression and secretion, as a result of downregulation of sPLA2-IIa promoter activity. More importantly, we demonstrated elevated serum sPLA2-IIa levels in prostate cancer patients. High serum sPLA2-IIa levels are associated significantly with high Gleason score and advanced disease stage. Increased sPLA2-IIa expression was confirmed in prostate cancer cells, but not in normal epithelium and stroma by immunohistochemistry analysis. We showed that elevated signaling of the HER/HER2-PI3K-Akt-NF-κB pathway contributes to sPLA2-IIa overexpression and secretion by prostate cancer cells. Given that sPLA2-IIa overexpression is associated with prostate development and progression, serum sPLA2-IIa may serve as a prognostic biomarker for prostate cancer and a potential surrogate prostate biomarker indicative of tumor burden.

Development of an electrode cell impedance method to measure osteoblast cell activity in magnesium-conditioned media.

Magnesium (Mg) as a biodegradable metal has potential advantages as an implant material. This paper studies the effect of magnesium ions on osteoblast (U2-OS) behavior since magnesium implants mainly dissolve as divalent magnesium ions (Mg(2+)). A real-time monitoring technique based on electric cell-substrate impedance sensing (ECIS) was used for measuring cell proliferation, migration, adhesion, and cytotoxicity in magnesium-conditioned media. The impedance results show that U2-OS proliferation and adhesion were inhibited in not only a magnesium-free medium but also in a medium with a high concentration of magnesium. The impedance method produced more sensitive results than the output of an MTT assay. Other standard bioanalytical tests were conducted for comparison with the ECIS method. Immunochemistry was carried out to study cell adhesion in magnesium-conditioned media by staining using F-actin and alpha-tubulin and correlated cell density on the electrode with impedance. Bone tissue formation was studied using von Kossa staining and indicated the mineralization level of cells in magnesium-conditioned media decreased with the increase of magnesium ion concentration. Real-time PCR provided gene expression indicators of cell growth, apoptosis, inflammation, and migration. Compared to the bioanalytical methods of immunochemistry and MTT assays, which need preparation time and post-washing step, ECIS was able to measure cell activity in real time without any cell culture modification. In summary, ECIS might be an effective way to study biodegradable magnesium implants.

Effect of Lithium and Aluminum on the Mechanical Properties, In Vivo and In Vitro Degradation, and Toxicity of Multiphase Ultrahigh Ductility Mg-Li-Al-Zn Quaternary Alloys for Vascular Stent Application.

Magnesium alloys are the most widely studied biodegradable metals for biodegradable vascular stent application. Two major issues with current magnesium alloy based stents are their low ductility and fast corrosion rates. Several studies have validated that introduction of Li into the magnesium alloys will significantly improve the ductility while alloying with Al will improve the corrosion resistance and strength. In the present study, we studied the effects of alloying different amounts of Li and Al on the Mg-Li-Al-Zn (LAZ) quaternary alloy system. Rods were made from four different LAZ alloys, namely, LAZ611, LAZ631, LAZ911, and LAZ931 following melting, casting, and then extrusion. Systematic assessment of mechanical properties, in vitro corrosion, cytotoxicity, and in vivo degradation including local and systemic toxicity conducted demonstrated the beneficial effects of Li and Al on the mechanical properties. Our results specifically suggest that alloying with Li significantly improved the ductility while Al enhanced the strength of the LAZ alloys. Four of the LAZ alloys exhibited different corrosion rates in Hank's balanced salt solution depending on the chemical composition. Indirect in vitro cytotoxicity tests also showed lower cytotoxicity for the alloys exhibiting higher corrosion resistance. In vivo corrosion rates in the mouse subcutaneous model showed different corrosion rates compared to the in vitro tests. Nevertheless, all of the four LAZ alloys displayed no local and systemic toxicity based on the histology analysis. This research study, therefore, demonstrated the benefits of using Li and Al as alloying elements in LAZ alloys and the potential use of LAZ alloys for vascular stent application.

Tiny medicine: nanomaterial-based biosensors.

Tiny medicine refers to the development of small easy to use devices that can help in the early diagnosis and treatment of disease. Early diagnosis is the key to successfully treating many diseases. Nanomaterial-based biosensors utilize the unique properties of biological and physical nanomaterials to recognize a target molecule and effect transduction of an electronic signal. In general, the advantages of nanomaterial-based biosensors are fast response, small size, high sensitivity, and portability compared to existing large electrodes and sensors. Systems integration is the core technology that enables tiny medicine. Integration of nanomaterials, microfluidics, automatic samplers, and transduction devices on a single chip provides many advantages for point of care devices such as biosensors. Biosensors are also being used as new analytical tools to study medicine. Thus this paper reviews how nanomaterials can be used to build biosensors and how these biosensors can help now and in the future to detect disease and monitor therapies.

Additive effects of a small molecular PCNA inhibitor PCNA-I1S and DNA damaging agents on growth inhibition and DNA damage in prostate and lung cancer cells.

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer.

Vav3 oncogene activates estrogen receptor and its overexpression may be involved in human breast cancer.

BACKGROUND: Our previous study revealed that Vav3 oncogene is overexpressed in human prostate cancer, activates androgen receptor, and stimulates growth in prostate cancer cells. The current study is to determine a potential role of Vav3 oncogene in human breast cancer and impact on estrogen receptor a (ERalpha)-mediated signaling axis. METHODS: Immunohistochemistry analysis was performed in 43 breast cancer specimens and western blot analysis was used for human breast cancer cell lines to determine the expression level of Vav3 protein. The impact of Vav3 on breast cancer cell growth was determined by siRNA knockdown of Vav3 expression. The role of Vav3 in ERalpha activation was examined in luciferase reporter assays. Deletion mutation analysis of Vav3 protein was performed to localize the functional domain involved in ERalpha activation. Finally, the interaction of Vav3 and ERalpha was assessed by GST pull-down analysis. RESULTS: We found that Vav3 was overexpressed in 81% of human breast cancer specimens, particularly in poorly differentiated lesions. Vav3 activated ERalpha partially via PI3K-Akt signaling and stimulated growth of breast cancer cells. Vav3 also potentiated EGF activity for cell growth and ERalpha activation in breast cancer cells. More interestingly, we found that Vav3 complexed with ERalpha. Consistent with its function for AR, the DH domain of Vav3 was essential for ERalpha activation. CONCLUSION: Vav3 oncogene is overexpressed in human breast cancer. Vav3 complexes with ERalpha and enhances ERalpha activity. These findings suggest that Vav3 overexpression may aberrantly enhance ERalpha-mediated signaling axis and play a role in breast cancer development and/or progression.

Smad3 is overexpressed in advanced human prostate cancer and necessary for progressive growth of prostate cancer cells in nude mice.

PURPOSE: The purpose of this study was to investigate the potential role of Smad3, a key mediator of transforming growth factor-beta signaling, in progression of prostate cancer. EXPERIMENTAL DESIGN: Expression of Smad proteins was determined in human prostate cancer tissue array and cell lines. Growth and metastasis of cells overexpressing dominant-negative Smad3 (Smad3D) were studied to determine its role in tumor progression in mice. Cell growth, apoptosis, and expression of angiogenic molecules in tumor lesions were studied to determine potential pathways that Smad3 promotes tumor progression. RESULTS: Smad3 was overexpressed in human prostate cancer, which correlated with Gleason score and expression of proliferating cell nuclear antigen. Androgen-independent PC-3MM2 and DU145 cells expressed much higher levels of Smad3 than did androgen-dependent LNCaP, 22Rv1, and LAPC-4 cells. Overexpression of Smad3D in PC-3MM2 cells (PC-3MM2-Smad3D) had minimal direct effects on cell growth but attenuated effects of transforming growth factor-beta1 on gene expression and cell growth. Overexpression of Smad3D did not significantly alter tumor incidence but reduced tumor growth rate and metastasis incidence. Most cells in the control tumors, but not PC-3MM2-Smad3D tumors, were positively stained by an antibody to proliferating cell nuclear antigen. Microvessels and expression of angiogenic molecule interleukin-8 were significantly reduced in tumors from PC-3MM2-Smad3D cells. PC-3MM2-Smad3D tumors also expressed lower levels of vascular endothelial growth factor and platelet-derived growth factor. CONCLUSIONS: These data suggest that Smad3, through regulating angiogenic molecule expression in tumor cells, is critical for progression of human prostate cancer.

A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells.

The purpose of this study was to determine the effects of 6-amino-2-[2-(4-tert-butyl-phenoxy)-ethylsulfonyl]-1H-pyrimidine-4-one (DL3), a novel synthetic compound with small-molecule drug properties, on androgen-regulated gene expression and cell growth in human prostate cancer cells. LNCaP, 22Rv1, and LAPC-4 cells were used in the studies. Expression of prostate-specific antigen (PSA) and androgen receptor (AR) was determined by ELISA, Western blotting, real-time reverse transcription-PCR, nuclear run-on, and/or promoter luciferase reporter assays. Effects of DL3 on cell growth were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining. DL3 inhibited dihydrotestosterone (DHT)-induced PSA expression in a dose-dependent fashion. The inhibitory effects of DL3 were more potent than those of flutamide, nilutamide, and bicalutamide. Moreover, DL3 blocked the stimulatory effects of nilutamide on PSA expression in LNCaP cells. Unlike the three classic antiandrogens, DL3 did not show intrinsic AR agonist activity. Nuclear run-on and PSA promoter reporter assays revealed that DL3 blocked DHT-induced PSA gene transcription. Consistent with its effects on PSA expression, DL3 inhibited DHT-stimulated cell growth with a potency significantly superior to flutamide, nilutamide, or bicalutamide. Furthermore, cells resistant to flutamide or nilutamide were as susceptible as their parental counterparts to the inhibitory effects of DL3 on both PSA expression and cell growth. DL3 did not inhibit AR nuclear localization and the NH(2)- and COOH-terminal interaction of AR induced by DHT. These data show that DL3 is a novel inhibitor of the AR signaling axis and a potentially potent therapeutic agent for the management of advanced human prostate cancer.

In Vitro and in Vivo Evaluation of Multiphase Ultrahigh Ductility Mg-Li-Zn Alloys for Cardiovascular Stent Application.

Magnesium alloys have been extensively studied as a novel biodegradable metallic material for cardiovascular stent application. However, the ductility limitation of magnesium alloy has been a key issue for biodegradable stents applications. In this study, two different multiphase ultrahigh ductility Mg-Li-Zn alloys, LZ61 and LZ91, are fabricated in the form of extruded rods and evaluated both in vitro and in vivo. The microstructure, mechanical properties and in vitro degradation are evaluated as well as in vitro cytotoxicity. The in vivo degradation, tissue response, and systematic toxicity are evaluated in a mouse subcutaneous model. Measurements show that LZ61 and LZ91 exhibit more than 40% elongation at fracture without significantly compromising the strength. Both in vitro and in vivo degradation showed low degradation rates for LZ61 but high degradation rate for the LZ91 alloy. Excellent biocompatibility is observed both in vivo and in vitro for LZ61 and LZ91. In summary, this study successfully demonstrates that the ultraductility multiphase Mg-Li-Zn alloy has the potential to be used for stent applications. Compared to LZ91, the LZ61 alloy shows better balance of mechanical properties, corrosion resistance, and biocompatibility, indicating its promise for cardiovascular stent applications.

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer.

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.

A carbon nanotube needle biosensor.

A carbon nanotube needle biosensor was developed to provide fast, cost effective and highly sensitive electrochemical detection of biomolecules. The sensor was fabricated based on an array of aligned multi-wall carbon nanotubes synthesized by chemical vapor deposition. A bundle of nanotubes in the array was welded onto the tip of a tungsten needle under a microscope. The needle was then encased in glass and a polymer coating leaving only the tip of the needle exposed. Cyclic voltammetry was performed to examine the redox behavior of the nanotube needle. The cyclic voltammetry results showed a steady-state response attributable to radial diffusion with a high steady-state current density. An amperometric sensor was then developed for glucose detection by physically attaching glucose oxidase on the nanotube needle. The amperometric response of these nanotube needles showed a high sensitivity with a low detection limit. It is expected that the nanotube needle can be sharpened to increase the sensitivity to the point where the current is almost too small to measure. The simple manufacturing method should allow commodity level production of highly sensitive electronic biosensors.

Carbon nanotubes grown on stainless steel to form plate and probe electrodes for chemical/biological sensing.

This paper describes the fabrication and evaluation of carbon nanotube (CNT) electrodes grown on stainless steel (SS) plate and wire for electrochemical sensor applications. Multi-wall carbon nanotubes with different diameters were grown on the SS plate and wire by chemical vapor deposition from an ethylene precursor. The SS provides a good electrical and mechanical connection to the CNT, and the SS is a tough substrate. The SS part of the electrode was electrically insulated from the analyte so that only the CNT were active in sensing. Cyclic voltammetry for the reduction of 6 mM K3Fe(CN)6 in a 1.0 M KNO3 supporting electrolyte was performed to examine the redox behavior of the CNT-SS electrode. The cyclic voltammograms showed sigmoidal-like shapes, indicating that mass transport around the electrodes is dominated by radial diffusion. Based on the cyclic voltammograms, the effective area of the CNT-SS electrodes and the number of individual CNTs were estimated. These results indicate that the CNT-SS plate and wire electrodes are good candidates to develop practical in vivo biosensors.