Thermal Stability and Orthogonal Functionalization of Organophosphonate Self-Assembled Monolayers as Potential Liners for Cu Interconnect.

In this study, we investigated the thermal stabilities of butylphosphonic acid (BPA) and aminopropyltriethoxysilane (APTES) self-assembled monolayers (SAM) on a Si substrate. The thermal desorption and the thermal cleavage of the BPA and APTES SAM film on the Si substrate were studied by X-ray photoelectron spectroscopy (XPS) upon thermal treatment from 50 to 550 °C. XPS analyses show that the onset of the thermal desorption of the APTES monolayer occurs at 250 °C and the APTES SAM completely decomposed at 400 °C. Conversely, BPA SAM on Si shows that the onset of thermal desorption occurs at 350 °C, and the BPA SAM completely desorbed at approximately 500 °C. Our study revealed that the organophosphonate SAM is a more stable SAM in modifying the dielectric sidewalls of a Cu interconnect when compared to organosilane SAM. To overcome the spontaneous reaction of the organophosphonate film on the metal substrate, a simple orthogonal functionalization method using thiolate SAM as a sacrificial layer was also demonstrated in this study.

Dynamics of antioxidant heme oxygenase-1 and pro-oxidant p66Shc in promoting advanced prostate cancer progression.

The castration-resistant (CR) prostate cancer (PCa) is lethal and is the second leading cause of cancer-related deaths in U.S. males. To develop effective treatments toward CR PCa, we investigated reactive oxygen species (ROS) signaling pathway for its role involving in CR PCa progression. ROS can regulate both cell growth and apoptosis: a moderate increase of ROS promotes proliferation; its substantial rise results in cell death. p66Shc protein can increase oxidant species production and its elevated level is associated with the androgen-independent (AI) phenotype of CR PCa cells; while heme oxygenase-1 (HO-1) is an antioxidant enzyme and elevated in a sub-group of metastatic PCa cells. In this study, our data revealed that HO-1 and p66Shc protein levels are co-elevated in various AI PCa cell lines as well as p66Shc cDNA-transfected cells. Knockdown and/or inhibition of either p66Shc or HO-1 protein leads to reduced tumorigenicity as well as a reduction of counterpart protein. Knockdown of HO-1 alone results in increased ROS levels, nucleotide and protein oxidation and induction of cell death. Together, our data indicate that elevated HO-1 protein levels protect PCa cells from otherwise apoptotic conditions induced by aberrant p66Shc/ROS production, which thereby promotes PCa progression to the CR phenotype. p66Shc and HO-1 can serve as functional targets for treating CR PCa.

Non-mitotic effect of albendazole triggers apoptosis of human leukemia cells via SIRT3/ROS/p38 MAPK/TTP axis-mediated TNF-α upregulation.

Albendazole (ABZ) is a microtubule-targeting anthelmintic that acts against a variety of human cancer cells, but the dependence of its cytotoxicity on non-mitotic effect remains elusive. Thus, we aimed to explore the mechanistic pathway underlying the cytotoxicity of ABZ in human leukemia U937 cells. ABZ-induced apoptosis of U937 cells was characterized by mitochondrial ROS generation, p38 MAPK activation, TNF-α upregulation and activation of the death receptor-mediated pathway. Meanwhile, ABZ induced tubulin depolymerization and G2/M cell cycle arrest. ABZ-induced SIRT3 degradation elicited ROS-mediated p38 MAPK activation, leading to pyruvate kinase M2-mediated tristetraprolin (TTP) degradation. Inhibition of TTP-mediated TNF-α mRNA decay elicited TNF-α upregulation in ABZ-treated cells. Either the overexpression of SIRT3 or abolishment of ROS/p38 MAPK activation suppressed TNF-α upregulation and rescued the viability of ABZ-treated cells. In contrast to the inhibition of ROS/p38 MAPK pathway, SIRT3 overexpression attenuated tubulin depolymerization and G2/M arrest in ABZ-treated cells. Treatment with a SIRT3 inhibitor induced TNF-α upregulation and cell death without the induction of G2/M arrest in U937 cells. Taken together, our data indicate that ABZ-induced SIRT3 downregulation promotes its microtubule-destabilizing effect, and that the non-mitotic effect of ABZ largely triggers apoptosis of U937 cells via SIRT3/ROS/p38 MAPK/TTP axis-mediated TNF-α upregulation. Notably, the same pathway is involved in the ABZ-induced death of HL-60 cells.

p66Shc regulates migration of castration-resistant prostate cancer cells.

Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

Factor VII-Induced MicroRNA-135a Inhibits Autophagy and Is Associated with Poor Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies worldwide. Treatment outcomes remain poor mainly due to lack of good diagnostic/prognostic markers and limited therapeutic strategies. We previously characterized aberrant activation of the TF/FVII/PAR2 pathway, which subsequently results in decreased autophagy, as a crucial event in malignant progression of HCC. Here, we identified miR-135a as a highly upregulated miRNA in HCC in response to TF/FVII/PAR2 activation. Analyzing 103 HCC patient specimens, we confirmed that miR-135a was frequently elevated in HCC tissues with higher FVII expression compared to adjacent non-cancerous counterparts. Increased miR-135a levels in HCC were also associated with tumor staging, recurrence, microvascular invasion, and decreased disease-free survival. We subsequently identified Atg14, a key component that regulates the formation of autophagosome as a direct target of miR-135a. Ectopic expression of miR-135a suppressed Atg14 levels and inhibited the autophagic processes. Our results indicate strong positive correlations between miR-135a levels and malignant behaviors in HCC patients and also suggest novel functions of miR-135a in regulation of autophagy, which could be useful as a potential target for prognostic and therapeutic uses.

Anti-Androgen Abiraterone Acetate Improves the Therapeutic Efficacy of Statins on Castration-Resistant Prostate Cancer Cells.

The treatment of castration-resistant (CR) prostate cancer (PCa) is limited. A sub-population of CR PCa tumors can synthesize androgens for intracrine androgen receptor (AR) activation, thus targeting androgen biosynthesis could be an effective therapeutic option for these patients. We determined that androgen biosynthesis inhibitors simvastatin, atorvastatin, and ketoconazole directly inhibit growth, migration, and colony formation of LNCaP C-81 cells, which exhibit de novo androgen biosynthesis, with simvastatin being the most effective. Importantly, in combination treatments, statins specifically enhanced growth suppression with added effects by anti-androgen abiraterone acetate on the CR PCa cells. Thus, statins can be used in conjunction with abiraterone acetate to enhance anti-androgen therapy for CR PCa.

Heteronemin Is a Novel c-Met/STAT3 Inhibitor Against Advanced Prostate Cancer Cells.

BACKGROUND: Prostate cancer is one of the most prevalent cancers in men worldwide. Aberrant activation of c-Met/signal transducer and activator of transcription-3 (STAT3) signaling is involved in prostate carcinogenesis, underscoring the demand for developing c-Met/STAT3-targeting drugs. Thus, we first utilized virtual screening strategy to identify STAT3-inhibiting marine compound, heteronemin, and then validated the STAT3-inhibiting function of heteronemin in prostate cancer cells. METHODS: Human prostate cancer LNCaP, DU145, and PC-3 cell lines were treated with heteronemin for 24 hr, then the cell viability was evaluated by MTT assay. Flow cytometry was performed to analyze the apoptosis in heteronemin-treated cells. Western blot and quantitative real-time PCR were executed to further confirm the c-Met/STAT3 signaling inhibition by heteronemin in DU145 and PC-3 cells. RESULTS: In this study, we employed the virtual screening strategy to identify heteronemin, a spongean sesterterpene, as a potential STAT3 inhibitor from Taiwan marine drugs library. Application of heteronemin potently suppressed the viability and anchorage-independent growth of human prostate cancer cells. Besides, heteronemin induced apoptosis in prostate cancer cells by activation of both intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways. By luciferase assay and expression analysis, it was confirmed that heteronemin inhibited the phosphorylation of c-Met/src/STAT3 signaling axis, STAT3-driven luciferase activities and expression of STAT3-regulated genes including Bcl-xL, Bcl-2, and Cyclin D1. Finally, heteronemin effectively antagonized the hepatocyte growth factor (HGF)-stimulated c-Met/STAT3 activation as well as the proliferation and colonies formation in refractory prostate cancer cells. CONCLUSIONS: These findings suggest that heteronemin may constitute a novel c-Met/STAT3-targeting agent for prostate cancer. Prostate 76:1469-1483, 2016. © 2016 Wiley Periodicals, Inc.

Design and synthesis of pyrrolobenzodiazepine-gallic hybrid agents as p53-dependent and -independent apoptogenic signaling in melanoma cells.

A new class of pyrrolo[2,1-c][1,4]benzodiazepine-Gallic hybrid agents (PBD-GA) conjugated through alkyl spacers has been designed and synthesized. The combination of these two core pharmacophores with modification in the C-8 position of the PBD ring with alkyl spacers afforded oxygen-tethered compounds 5a-5d and amide-tethered analogues 11a-11d with improved anticancer activity for two melanoma cell lines, A375 and RPMI7951, differing in their p53 status. The agents 5a-5d were cytotoxic in melanoma compared to agents 11a-11d. In particular, compounds 5b and 5c were found to possess the most potent activity compared with other hybrid agents and were proved with the help of quantitative structure activity relationship studies (QSAR). These PBD conjugates caused S phase arrest for the A375 cell line via increased reactive oxygen species (ROS) generation, deoxyribonucleic acid (DNA) damage, ataxia telangiectasia mutated (ATM)/ATM-Rad3-related (ATR) and checkpoint kinases 1 (Chk1) activation. Moreover, the PBD-GA induced A375 apoptotic cell death followed through p53 (ATM downstream target) increase, B-cell leukemia-xL (Bcl-xL) and mitochondrial membrane potential (ΔΨmt) decrease, cytochrome c release, and caspase-3/Poly Adp Ribose Polymerase (PARP) cleavage. On the other hand, mutant p53 RPMI7951 cell death occurred by PBD-GA-mediated mitochondria- and caspase-dependent pathways via lysosomal membrane permeabilization (LMP), but not through p53 signaling. Finally, compound 5b was shown to reduce murine melanoma size in a mouse model. These results suggest that the PBD-GA could be used as a useful chemotherapeutic agent in melanoma with activated p53 or mutant p53.

Cellular prostatic acid phosphatase (cPAcP) serves as a useful biomarker of histone deacetylase (HDAC) inhibitors in prostate cancer cell growth suppression.

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

Discovery of Novel N-alkyl 4-anilinofuro[2,3-b]quinoline Derivatives (CIL-102 Derivatives) Against Castration-resistant Human Prostate Cancers.

A number of N-alkylated 4-anilinofuro[2,3-b]quinoline derivatives were synthesized and evaluated in vitro against PC-3, A549, and MCF-7 cancer cells and M-10 normal human mammary epithelial cells. The known antimitotic CIL-102 was moderately active against the growth of PC-3 prostate cancer cells with an IC50 value of 2.69 µM while it was more potent against the growth of A549, MCF-7 and M-10 cells with IC50 values of 0.61, 0.31 and 0.95 µM, respectively. However, the cytotoxic profiles of its N-alkylated derivatives, 6a - 6c, were reversed and strongly inhibited PC-3 cell growth with IC50 values of less than 1.0 µM but only weakly against the growth of A549, MCF-7 and M-10 cells. These results indicated that N-alkylation of CIL-102 increased not only selectivity but also the antiproliferative potency against PC-3 cell growth. Among these derivatives synthesized, N-(4-acetylphenyl)-N-(furo[2,3-b]quinolin- 4-yl)methylamine (6a) and its N-ethyl counterpart 6b are the two most active CIL-102 derivatives against PC-3 cell growth with IC50 value of 0.22 and 0.20 µM, respectively. Compound 6a is less cytotoxic to normal human M-10 cells than 6b and therefore was selected for further mechanism studies. The flow cytometry studies clearly indicated that compound 6a induced cell accumulation in G2/M phase in a dose-dependent manner after 24 h-treatment. While the proliferation of LNCaP C-81 prostate cancer cells was also strongly suppressed by compound 6a; compound 11a exhibited better selective activity toward LNCaP C-81 prostate cancer cells over RWPE-1 non-cancerous prostate epithelia. Thus, this group of compounds has a potential of serving as therapeutic agents toward advanced castration-resistant prostate cancers.

p66Shc longevity protein regulates the proliferation of human ovarian cancer cells.

p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression.

Antiproliferative activity of novel imidazopyridine derivatives on castration-resistant human prostate cancer cells.

Metastatic prostate cancer (mPCa) relapses after a short period of androgen deprivation therapy and becomes the castration-resistant prostate cancer (CR PCa); to which the treatment is limited. Hence, it is imperative to identify novel therapeutic agents towards this patient population. In the present study, antiproliferative activities of novel imidazopyridines were compared. Among three derivatives, PHE, AMD and AMN, examined, AMD showed the highest inhibitory activity on LNCaP C-81 cell proliferation, following dose- and time-dependent manner. Additionally, AMD exhibited significant antiproliferative effect against a panel of PCa cells, but not normal prostate epithelial cells. Further, when compared to AMD, its derivative DME showed higher inhibitory activities on PCa cell proliferation, clonogenic potential and in vitro tumorigenicity. The inhibitory activity was apparently in part due to the induction of apoptosis. Mechanistic studies indicate that AMD and DME treatments inhibited both AR and PI3K/Akt signaling. The results suggest that better understanding of inhibitory mechanisms of AMD and DME could help design novel therapeutic agents for improving the treatment of CR PCa.

Apoptosis induced by 2-aryl benzothiazoles-mediated photodynamic therapy in melanomas via mitochondrial dysfunction.

A mild and efficient synthetic development of 2-arylbenzothiazoles 5 mediated by ceric ammonium nitrate (CAN) via intramolecular cyclization of N-phenyl-thiobenzamides 4 was achieved. Further compounds 5 were reduced to corresponding amines 6, and their photodynamic therapy (PDT) effect was evaluated on malignant human melanoma A375 cells. Amine 6l plus ultraviolet A (UVA) induced caspase-3 activity, poly(ADP-ribose)polymerase cleavage, M30 positive CytoDeath staining, and subsequent apoptotic cell death. Our data disclosed that treatment of A375 cells with 6l plus UVA resulted in a decrease in mitochondrial membrane potential (ΔΨmt), oxidative phosphorylation system (OXPHOS) subunits, and adenosine triphosphate (ATP) but an increase in mitochondrial DNA 4977-bp deletion via reactive oxygen species (ROS) generation. Transmission electron microscopy (TEM) observations also showed major ultrastructural alterations of mitochondria. Additionally, 6l plus UVA was also shown to reduce murine melanoma size in a mouse model. The present study supports the hypothesis that 6l-PDT may serve as a potential ancillary modality for the treatment of melanoma.

Validation of the effects of TGF-ß1 on tumor recurrence and prognosis through tumor retrieval and cell mechanical properties.

BACKGROUND: In vivo, the transforming growth factor-beta1 (TGF-ß1)-induced epithelial to mesenchymal transition (EMT) occurs in seconds during cancer cells intravasation and extravasation. Although it has been established that cellular stiffness can change as a cancer cell transformed, the precise relationship between TGF-ß1-induced mesenchymal stem cell mechanics and cancer prognosis remains unclear. Accordingly, it is hard to define the effects of EMT on cell mechanical properties (CMs), tumor recurrence and metastasis risks. This study bridges physical and pathological disciplines to reconcile single-cell mechanical measurements of tumor cells. METHODS AND RESULTS: We developed a microplate measurement system (MMS) and revealed the intrinsic divergent tumor composition of retrieval cells by cell stiffness and adhesion force and flow cytometry analysis. After flow cytometry sorting, we could measure the differences in CMs of the Sca-1+-CD44+ (mesenchymal-stem-cell-type) and the other subgroups. As well as the stiffer and heterogeneous compositions among tumor tissues with higher recurrence risk were depicted by MMS and atomic force microscopy (AFM). An in vitro experiment validated that Lewis lung carcinoma (LLC) cells acquired higher CMs and motility after EMT, but abrogated by SB-505124 inhibition. Concomitantly, the CD31, MMP13 and TGF-ß1 enriched micro-environment in the tumor was associated with higher recurrence and distal lung metastasis risks. Furthermore, we report a comprehensive effort to correlate CMs to tumor-prognosis indicators, in which a decreased body weight gain ratio (BWG) and increased tumor weight (TW) were correlated with increased CMs. CONCLUSIONS: Together, we determined that TGF-ß1 was significantly associated with malignant tumor progressing. In terms of clinical applications, local tumor excision followed by MMS analysis offers an opportunity to predict tumor recurrence and metastasis risks.

Ran GTPase-activating protein 1 is a therapeutic target in diffuse large B-cell lymphoma.

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1(+), while reactive lymphoid hyperplasia (n = 12) was RanGAP1(+) predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin's lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.

Discovery, synthetic methodology, and biological evaluation for antiphotoaging activity of bicyclic[1,2,3]triazoles: in vitro and in vivo studies.

Novel bicyclic[1,2,3]triazoles (4, 7, 11, 15) have been synthesized using a one-pot metal free strategy with high structural diversity as photoprotective agents, and their effect on UVA-induced senescence in human dermal fibroblast cells (FB) and the associated mechanism are delineated. 11d plus UVA can induce a decrease in reactive oxygen species (ROS) production and senescence-associated ß-galactosidase (SA-ß-gal) activity but an increase in adenosine triphosphate (ATP) synthesis and mitochondrial membrane potential (ΔΨmt). The mRNA levels of six senescence-associated genes, matrix metalloproteinase-1 (MMP-1), was decreased, while elastin, procollagen I type I, fibronectin, COL1α1, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were increased. 11d plus UVA also decreased MMP-1 and increased TIMP-1 protein levels. Additionally, the thickness of the murine dorsal skin and epidermis, by UVA, was decreased by topical 11d treatment. Our results indicate that bicyclic[1,2,3]triazoles protect UVA-induced senescence-like characteristics in FB cells, which may provide potential prevention against photoaging.

Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.

Quantification of ovarian cancer markers with integrated microfluidic concentration gradient and imaging nanohole surface plasmon resonance.

Nanohole array-based biosensors integrated with a microfluidic concentration gradient generator were used for imaging detection and quantification of ovarian cancer markers. Calibration curves based on controlled concentrations of the analyte were created using a microfluidic stepped diffusive mixing scheme. Quantification of samples with unknown concentration of analyte was achieved by image-intensity comparison with the calibration curves. The biosensors were first used to detect the immobilization of ovarian cancer marker antibodies, and subsequently applied for the quantification of the ovarian cancer marker r-PAX8 (with a limit of detection of about 5 nM and a dynamic range from 0.25 to 9.0 µg.mL(-1)). The proposed biosensor demonstrated the ability of self-generating calibration curves on-chip in an integrated microfluidic platform, representing a further step towards the development of comprehensive lab-on-chip biomedical diagnostics based on nanohole array technology.

Pharmacophore-based virtual screening and biological evaluation of small molecule inhibitors for protein arginine methylation.

Protein arginine methyltransferases (PRMTs) are proved to play vital roles in chromatin remodeling, RNA metabolism, and signal transduction. Aberrant regulation of PRMT activity is associated with various pathological states such as cancer and cardiovascular disorders. Development and application of small molecule PRMT inhibitors will provide new avenues for therapeutic discovery. The combination of pharmacophore-based virtual screening methods with radioactive methylation assays provided six hits identified as inhibitors against the predominant arginine methyltransferase PRMT1 within micromolar potency. Two potent compounds, A9 and A36, exhibited the inhibitory effect by directly targeting substrate H4 other than PRMT1 and displayed even higher inhibition activity than the well-known PRMT inhibitors AMI-1. A9 significantly inhibits proliferation of castrate-resistant prostate cancer cells. Together, A9 may be a potential inhibitor against advanced hormone-independent cancers, and the work will provide clues for the future development of specific compounds that block the interaction of PRMTs with their targets.

Reactive oxygen species induced by p66Shc longevity protein mediate nongenomic androgen action via tyrosine phosphorylation signaling to enhance tumorigenicity of prostate cancer cells.

Steroid hormones exhibit diverse biological activities. Despite intensive studies on steroid function at the genomic level, their nongenomic actions remain an enigma. In this study, we investigated the role of reactive oxygen species (ROS) in androgen-stimulated prostate cancer (PCa) cell proliferation. In androgen-treated PCa cells, increased cell growth and ROS production correlated with elevated p66Shc protein, an authentic oxidase. This growth stimulation was blocked by antioxidants. Further, elevated expression of p66Shc protein by cDNA transfection encoding wild-type protein, but not a redox-deficient (W134F) mutant, was associated with increased PCa cell proliferation. Conversely, knockdown of p66Shc expression by shRNA resulted in diminished cell growth. Increased p66Shc expression in PCa cells enhanced their tumorigenicity in xenograft animals. Importantly, p66Shc protein level is higher in clinical prostate adenocarcinomas than in adjacent noncancerous cells. Expression of redox-deficient p66Shc mutant protein abolished androgen-stimulated cell growth. In androgen-treated, H(2)O(2)-treated, and p66Shc cDNA-transfected PCa cells, cellular prostatic acid phosphatase, an authentic tyrosine phosphatase, was inactivated by reversible oxidation; subsequently, ErbB-2 was activated by phosphorylation at tyrosine-1221/1222. These results together support the notion that androgens induce ROS production through the elevation of p66Shc protein, which inactivates tyrosine phosphatase activity for the activation of interacting tyrosine kinase, leading to increased cell proliferation and enhanced tumorigenicity. Our results thus suggest that p66Shc protein functions at the critical junction point between androgens and tyrosine phosphorylation signaling in human PCa cells.