nitrophenols and Prostatic-Neoplasms

In many tumors, including prostate cancer, anti-apoptotic members of the Bcl-2 family are overexpressed and cause cell death resistance, which is a typical hallmark of cancer. Different therapeutic approaches, therefore, aim to restore the death mechanisms for enhanced apoptosis. Our recombinant immunotoxin D7(VL-VH)-PE40 is composed of the scFv D7(VL-VH) against the prostate-specific membrane antigen (PSMA) on the surface of prostate cancer cells and of the cytotoxic domain of the bacterial toxin Pseudomonas Exotoxin A (PE40). Since Pseudomonas Exotoxin A-based immunotoxins are known to preferentially inhibit the expression of the anti-apoptotic protein Mcl-1, the rationale was to test our immunotoxin in combination with the BH3 mimetic ABT-737, which specifically inhibits Bcl-2, Bcl-xl, and Bcl-w for enhanced induction of apoptosis in prostate cancer cells. The immunotoxin showed high and specific binding and cytotoxicity against PSMA expressing prostate cancer cells marked by a direct inhibition of Mcl-1. The combination of the immunotoxin with a subtoxic concentration of ABT-737 caused additive or even synergistic effects, which were based on an enhanced apoptosis induction as detected by poly(ADP-ribose) polymerase (PARP) and Caspase-3 cleavage in Western blot. Our study shows that the combination therapy of immunotoxin plus ABT-737 is a promising approach for the future treatment of advanced prostate cancer to improve therapeutic efficacy and to reduce adverse side effects.

Topics: ADP Ribose Transferases; Apoptosis; Bacterial Toxins; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; Exotoxins; Humans; Immunotoxins; Male; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Peptide Fragments; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins; Pseudomonas aeruginosa Exotoxin A; Sulfonamides; Virulence Factors

In this study, Broccoli green extract was reported as a green and environmental friendly precursor for the one-pot biosynthesis of copper nanoparticles. The synthesized nanoparticles were characterized by UV-vis, FTIR, TEM, DLS, XRD and cyclic voltammetry. The TEM and DLS results showed that the NPs are in spherical and monodispersed with an average particle size of ~4.8nm. The FTIR results confirmed the occurrence of bioactive functional groups that are responsible for reducing cupric sulphate to copper ions. The UV-vis spectrophotometry was used for catalytic reduction of 4-nitrophenol and its dynamic reaction in Britton-Robinson buffer solution. This catalytic activity was further supported with methylene blue and methyl red dyes degradation. The nanocatalyst can be recovered from the reaction mixture and reused many times with none vital loss of catalytic activity. The Broccoli green extract modified copper nanoparticles coated on screen printing electrode laid a new sensing platform and has an excellent electrocatalytic activity. Furthermore, surface modified CuNPs with Broccoli green extract exhibited no cytotoxicity at the concentration ranging from 0.5 to 1.5μM on the prostate cancer (PC-3) cell lines. The maximum scavenging % of Broccoli green extract modified CuNPs was found to be >70.50% at the concentration of 0.25mM against 1,1-diphenyl-2-picrylhydrazyl.

Topics: Antioxidants; Brassica; Catalysis; Cell Line, Tumor; Cell Survival; Copper; Dynamic Light Scattering; Electrochemical Techniques; Green Chemistry Technology; Humans; Male; Metal Nanoparticles; Methylene Blue; Microscopy, Electron, Transmission; Nitrophenols; Plant Extracts; Prostatic Neoplasms; Spectroscopy, Fourier Transform Infrared

Semiconductor quantum dots (QDs), after surface modification to provide water solubility and biocompatibility, have a promising future in biomedical applications. In this study, a dual receptor-targeting dual-modality PET/near-infrared fluorescence (NIRF) probe was developed for accurate assessment of the pharmacokinetics and tumor-targeting efficacy of QDs.. QDs were modified by β-Glu-RGD-BBN (RGD is arginine-glycine-aspartate acid, and BBN is bombesin) peptides and then labeled with (18)F via the 4-nitrophenyl-2-(18)F-fluoropropionate prosthetic group. Cytotoxicity and cell-binding assay of QD-RGD-BBN were performed with PC-3 cells. In vivo dual-modality PET/NIRF imaging of prostate tumor-bearing mice was investigated using QD-RGD-BBN and 2-(18)F-fluoropropionyl-QD-RGD-BBN ((18)F-FP-QD-RGD-BBN). An in vivo biodistribution study of (18)F-FP-QD-RGD-BBN was performed on normal mice.. QD-RGD-BBN exhibited strong red luminescence (600-800 nm) with the same maximum fluorescence wavelength (705 nm) as QD705 and slightly lower toxicity than that of QD705 in PC-3 cells at concentrations of greater than 30 μg/mL. Uptake of QD-RGD-BBN in PC-3 cells showed no significant decrease in the presence of an excess amount of dimer arginine-glycine-aspartate acid (RGD2) or bombesin(7-14) (BBN) peptide but was blocked significantly in the presence of an excess amount of NH2-RGD-BBN. Dual-function PET/NIRF imaging is able to accurately assess the biodistribution and tumor-targeting efficacy of the (18)F-labeled functionalized QDs.. The functionalized QD probe has great potential as a universal dual-targeting probe for detecting tumors in living subjects, opening up a new strategy for the development of multitargeting multimodality (18)F-labeled QD probes with improved tumor-targeting efficacy.

Topics: Animals; Biocompatible Materials; Bombesin; Copper Radioisotopes; Female; Male; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms; Nitrophenols; Oligopeptides; Peptides; Positron-Emission Tomography; Prostatic Neoplasms; Quantum Dots; Receptors, Bombesin; Semiconductors; Spectrometry, Fluorescence; Tissue Distribution

Docetaxel (DTX) is a useful chemotherapeutic drug for the treatment of hormone-refractory prostate cancer. However, emergence of DTX resistance has been a therapeutic hurdle. In this study, we investigated the effect of combining DTX with Bcl-2 family inhibitors using human prostate cancer cell lines (PC3, LNCaP, and DU145 cells). PC3 cells were less sensitive to DTX than were the other two cell lines. In contrast to ABT-199, which inhibits Bcl-2 and Bcl-w, both ABT-263 and ABT-737, which inhibit Bcl-2, Bcl-xL, and Bcl-w, significantly augmented the antitumor effect of DTX on PC3 cells. ABT-263 also enhanced the antitumor effect of DTX on a DTX-resistant PC3 variant cell line. The antitumor effect of ABT-263 was due mainly to its inhibitory effect on Bcl-xL. In a xenograft mouse model, DTX and ABT-737 combination therapy significantly inhibited PC3 tumor growth. Interestingly, although ABT-263 activated caspase-9 in PC3 cells, inhibition of caspase-9 unexpectedly promoted ABT-263-induced apoptosis in a caspase-8-dependent manner. This augmented apoptosis was also observed in LNCaP cells. These findings indicate that Bcl-xL inhibition can sensitize DTX-resistant prostate cancer cells to DTX, and they reveal a unique apoptotic pathway in which antagonism of Bcl-2 family members in caspase-9-inhibited prostate cancer cells triggers caspase-8-dependent apoptosis.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Docetaxel; Drug Synergism; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nitrophenols; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Taxoids; Transfection; Xenograft Model Antitumor Assays

Pim serine/threonine kinases contribute to prostate tumorigenesis and therapeutic resistance, yet Pim kinase inhibitors seem to have only limited effects on prostate cancer cell survival. Because overexpression of Bcl-2 family members are implicated in chemotherapeutic resistance in prostate cancer, we investigated the cooperative effects of Pim kinase inhibition with ABT-737, a small molecule antagonist of Bcl-2 family members. Strikingly, the addition of ABT-737 to Pim inhibitors triggered a robust apoptosis of prostate cancer cells in vitro and in vivo. Pim inhibitors decreased levels of the Bcl-2 family member Mcl-1, both by blocking 5'-cap dependent translation and decreasing protein half life. In addition, Pim inhibition transcriptionally increased levels of the BH3 protein Noxa by activating the unfolded protein response (UPR), lead to eIF-2α phosphorylation and increased expression of CHOP. Increased levels of Noxa also inactivated the remaining levels of Mcl-1 protein activity. Notably, these specific protein changes were essential to the apoptotic process because ABT-737 did not inhibit Mcl-1 protein activity and Mcl-1 overexpression blocked the apoptotic activity of ABT-737. Our results therefore suggest that this combination treatment could be developed as a potential therapy for human prostate cancer where overexpression of Pim kinases and antiapoptotic Bcl-2 family members drives tumor cell resistance to current anticancer therapies.

Topics: Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Endoplasmic Reticulum; Humans; Male; Nitrophenols; Oxidative Stress; Piperazines; Prostatic Neoplasms; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-pim-1; Sulfonamides

Targeting multiple anti-apoptotic proteins is now possible with the small molecule BH3 domain mimetics such as ABT-737. Given recent studies demonstrating that autophagy is a resistance mechanism to multiple therapeutic agents in the setting of apoptotic inhibition, we hypothesized that hydroxychloroquine (HCQ), an anti-malarial drug that inhibits autophagy, will increase cytotoxicity of ABT-737.. Cytotoxicity of ABT-737 and HCQ was assessed in vitro in PC-3 and LNCaP cells, and in vivo in a xenograft mouse model. The role of autophagy as a resistance mechanism was assessed by siRNA knockdown of the essential autophagy gene beclin1. ROS was measured by flow cytometry, and mitophagy assessed by the mCherry-Parkin reporter.. Induction of autophagy by ABT-737 was a mechanism of resistance in prostate cancer cell lines. Therapeutic inhibition of autophagy with HCQ increased cytotoxicity of ABT-737 both in vitro and in vivo. ABT-737 induced LC-3 and decreased p62 expression by immunoblot in cell lines and by immunohistochemistry in tumors in vivo. Assessment of ROS and mitochondria demonstrated that ROS production by ABT-737 and HCQ was a mechanism of cytotoxicity.. We demonstrated that autophagy inhibition with HCQ enhances ABT-737 cytotoxicity in vitro and in vivo, that LC-3 and p62 represent assessable markers in human tissue for future clinical trials, and that ROS induction is a mechanism of cytotoxicity. These results support a new paradigm of dual targeting of apoptosis and autophagy in future clinical studies.

Topics: Animals; Apoptosis; Autophagy; Biphenyl Compounds; Cell Line, Tumor; Drug Therapy, Combination; Humans; Hydroxychloroquine; Male; Mice; Mice, Nude; Nitrophenols; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Treatment Outcome; Xenograft Model Antitumor Assays

To investigate the synergistical killing effect of docetaxel combined with ABT-737 on human prostate cancer cell line PC-3 by inducing apoptosis and further to determine the mechanism underlying such effect.. PC-3 cells were treated with various concentrations of docetaxel or (and) ABT-737. Cell viability was determined using MTT assay. Apoptosis was assessed by fluorescence microscopy analysis of cells with condensed and segmented nuclei following staining with 4',6-diamidino-2-phenylindole (DAPI). Cellular DNA was stained with propidium iodide and flow cytometric analysis was performed to analyze the cell cycle distribution. Bcl-2, Bax, Bcl-xL and Mcl-1 protein changes were detected by Western blot. The activity of caspase-3 was measured using a colorimetric assay.. Docetaxel (20 nmol/L) combination with ABT-737 (400 nmol/L) for 48 hours, the cell viability was decreased to 19.7% ± 3.2% to compare with 44.2% ± 4.4% (t = 4.45) of docetaxel and 93.2% ± 1.8% of ABT-737 separately and there was a synergistic effect between the two drugs (CI = 0.8). Apoptosis rate of the combination group was higher than other two drugs. Docetaxel increased the cell number arrested in G(2)/M phase compared with control group (P < 0.05), but the combination treatment resulted in a significant arrest in the G(0)/G(1) phase. The combination treatment could significantly reduced the Bcl-2, Bcl-xL and Mcl-1 expression (F = 369.53, 57.89 and 32.77, all P < 0.05) and enhanced the activity of caspase-3 (419.7% ± 15.6%) (F = 207.33, P < 0.05).. The combination of ABT-737 with docetaxel can synergistically inhibit the proliferation of PC-3 cells through inducing apoptosis, which may be associated with cell cycle arrest, down-regulation of Bcl-2, Bcl-xL and Mcl-1 expression and activation of caspase-3.

Topics: Apoptosis; bcl-X Protein; Biphenyl Compounds; Caspase 3; Cell Cycle; Cell Line, Tumor; Docetaxel; Drug Synergism; Humans; Male; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Taxoids

We report a novel continuous and sensitive fluorescence turn-on assay for ACPs, which consists of a cationic conjugated polyelectrolyte (PPE4+) and a commonly used phosphatase substrate p-nitrophenyl phosphate (pNPP). The kinetics of the ACP catalyzed hydrolysis of the substrate pNPP was monitored by the fluorescence change of PPE4+ and corresponding kinetic parameters were derived to be consistent with the literature reports. The applications of PPE4+/pNPP-based ACP assay in high-throughput screening of ACP inhibitors and detection of prostatic acid phosphotase (PAP) in vitro were demonstrated.

Topics: Acid Phosphatase; Aniline Compounds; Catalysis; Drug Design; Electrolytes; Humans; Hydrolysis; Kinetics; Male; Models, Chemical; Nitrophenols; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Polymers; Prostatic Neoplasms; Protein Tyrosine Phosphatases; Spectrometry, Fluorescence

Apoptosis resistance is a hallmark of cancer linked to disease progression and treatment resistance, which has led to the development of anticancer therapeutics that restore apoptotic function. Antiapoptotic Bcl-2 is frequently overexpressed in refractory prostate cancer and increased following standard hormonal therapy and chemotherapy; however, the rationally designed Bcl-2 antagonist, ABT-737, has not shown single agent apoptosis-promoting activity against human prostate cancer cell lines. This is likely due to the coordinate expression of antiapoptotic, Bcl-2-related Mcl-1 that is not targeted by ABT-737. We developed a mouse model for prostate cancer in which apoptosis resistance and tumorigenesis were conferred by Bcl-2 expression. Combining ABT-737 with agents that target Mcl-1 sensitized prostate cancer cell lines with an apoptotic block to cell death in vitro. In mice in vivo, ABT-737 showed single agent efficacy in prostate tumor allografts in which tumor cells are under hypoxic stress. In human prostate cancer tissue, examined using a novel tumor explant system designated Tumor Tissue Assessment for Response to Chemotherapy, combination chemotherapy promoted efficient apoptosis. Thus, rational targeting of both the Bcl-2 and Mcl-1 mechanisms of apoptosis resistance may be therapeutically advantageous for advanced prostate cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Cisplatin; Drug Synergism; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Paclitaxel; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Suppressor Protein p53

Gene therapy shows promise for treating prostate cancer and has been evaluated in several clinical trials. A major challenge that remains is to establish a method for verifying transgene activity in situ. The lacZ gene encoding beta-galactosidase historically has been the most popular reporter gene for molecular biology. We have designed a 19F NMR approach to reveal lacZ gene expression by assessing beta-galactosidase (beta-gal) activity in vivo. The substrate 2-fluoro-4-nitrophenyl beta-D-galactopyranoside (OFPNPG) is readily hydrolyzed by beta-gal with a corresponding decrease in the 19F-NMR signal from OFPNPG and the appearance of a new signal shifted 4-6 ppm upfield from the aglycone 2-fluoro-4-nitrophenol (OFPNP). We report proof of principle in cultures of PC3 prostate cancer cells using 19F NMR spectroscopy and 19F chemical shift imaging. More importantly, we demonstrate for the first time the ability to differentiate wild-type and lacZ-expressing prostate tumor xenografts in mice using this approach.

Topics: Adenocarcinoma; Animals; beta-Galactosidase; Cell Line, Tumor; Enzyme Induction; Fluorine; Genes, Reporter; Genetic Therapy; Humans; Hydrolysis; Lac Operon; Magnetic Resonance Spectroscopy; Male; Mice; Nitrophenols; Nitrophenylgalactosides; Prostatic Neoplasms; Recombinant Fusion Proteins; Substrate Specificity; Transfection; Transplantation, Heterologous

A capture immunoenzyme assay (CIEA) for prostatic acid phosphatase (PAP) was developed and used to study the stability of this isoenzyme. Immunospecifically purified goat antibodies to PAP were covalently bound to special discs and used to capture the enzyme in serum samples in a weakly acidic medium during the first incubation (2 h) at 37 degrees C. The capture enzyme was then measured by its catalytic activity with p-nitrophenyl phosphate as substrate during the second incubation (1 h) at 37 degrees C. As much as 98% of the PAP in test specimens was captured and measured by this CIEA. The test results were expressed as enzymatic activity (U/l), extrapolated from a standard curve which was linear between 0.026 and 70 U/l. In test sera stored at 4 degrees C, the PAP was variably stable for 7 to 70 days, but the enzyme was quite stable in serum when stored at -20 degrees C for at least 156 days. At room temperature, when the sera were appropriately acidified, there was no loss of enzymatic activity for periods of 15 days, and in some cases, a large proportion of activity was still intact after 70 days. At 4 degrees C, as well as -20 degrees C, acidified serum and the partially purified PAP standard showed complete stability for at least 7 months. The CIEA reactivity of positive test specimens was inhibited by L(+)-tartaric acid, but not by cupric sulfate. The acid phosphatases of blood cell extracts were non-reactive in the CIEA procedure. The CIEA results of 224 serum samples from patients with and without prostate cancer correlated very well with those obtained by two direct enzymatic and two commercial RIA procedures, with correlation coefficients between 0.960 and 0.993, and diagnostic agreement between 86% and 100%.

Topics: Acid Phosphatase; Adult; Cold Temperature; Drug Stability; Female; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Male; Neoplasms; Nitrophenols; Organophosphorus Compounds; Prostate; Prostatic Neoplasms; Serum Albumin, Bovine; Time Factors

Topics: Acid Phosphatase; Clinical Enzyme Tests; Humans; Isoenzymes; Male; Naphthalenes; Nitrophenols; Organophosphorus Compounds; Prostate; Prostatic Neoplasms; Spectrophotometry, Ultraviolet; Tartrates; Thymolphthalein; United States

Measurements of serum prostatic acid phosphatase concentrations (PAP) by radioimmunoassay (RIA) were compared with the conventional measurements of serum acid phosphatase activities using p-nitrophenylphosphate (pNPP, tot.) and magnesium thymolphthalein monophosphate (TMP) as substrates and L(+)-tartrate (pNPP, tr.) as inhibitor in five prostatic cancer patients before therapy and in 13 during therapy. Elevated serum acid phosphatase activities were detected in 2, 2 and 3 of the 5 untreated patients when using pNPP (tot.), pNPP (tr.) and TMP enzyme assays, respectively. RIA for PAP detected elevated concentrations of the enzyme in 4 of these patients' sera. Three of the patients without metastases and one patient with suspected metastases had elevated concentrations of PAP by RIA. Serum acid phosphatase isoenzyme 2, which is mainly of prostatic origin, was separated chromatographically from serum samples with increased acid phosphatase activity. It represented 60--92% of the total activity, when TMP was used as substrate. Significant correlations (beta less than 0.001) were observed between all conventional enzyme activity measurements used and PAP by RIA within the whole patient group (n = 18), but no correlations existed within the patient group (p = 6) of high normal, or low abnormal serum PAP (2.7--6.6 micrograms/l). In addition, PAP measured by RIA better reflected the clinical state of the 13 patients under treatment than the conventional enzyme assays investigated.

Topics: Acid Phosphatase; Humans; Isoenzymes; Male; Nitrophenols; Organophosphorus Compounds; Prostatic Neoplasms; Radioimmunoassay; Thymolphthalein

Potent inhibition of human prostatic carcinoma tissue acid phosphatase by N,N-d-di-2-chloroethylaminophenol (AMOH) and N,N-p-di-2-chloroethylaminophenyl phosphate (AMPh) is described. Certain other difunctional nitrogen mustards were also inhibitory but N,N-p-di-2hydroxyethylaminophenol, the non-alkylating fully hydrolysed product from AMOH, was not. Inactivation of the enzyme by AMPh was progressive with time, showed apparent first order reaction kinetics and was not reversed by extensive dialysis. The results suggest that the inability of the enzyme to catalyse the hydrolysis of AMPh was due to inhibition in the presence of AMPh, possibly involving an alkylating mechanism. The implications for possible chemotherapy with AMPh are discussed.

Topics: Acid Phosphatase; Aniline Mustard; Humans; In Vitro Techniques; Kinetics; Male; Nitrogen Mustard Compounds; Nitrophenols; Organophosphorus Compounds; Prostatic Neoplasms; Substrate Specificity

The sensitivity of a recently developed solid phase radioimmunoassay for human prostatic acid phosphatase was compared to that of an enzymatic method using p-nitrophenylphosphate as substrate. In 109 histologically verified untreated stages I to IV prostatic cancers and 200 men without such cancer the solid phase radioimmunoassay method demonstrated substantially greater sensitivity and specificity than the enzymatic technique. In the 109 prostatic malignancies the immunochemical method correctly classified 80 (73 per cent) versus 34 (31 per cent) for the p-nitrophenylphosphate enzymatic technique (p less than 10(-6). In 44 stages I and II cancers confined to the prostate the radioimmunoassay was abnormally elevated in 19 (43 per cent) with only 4 (9.1 per cent) enzymatic elevations (p less than 10(-3). In 65 stages III and IV extraprostatic cancers correct classifications were noted in 61 (94 per cent) of the radioimmunoassays and 30 (46 per cent) enzymatic tests (p less than 10(-6). The radioimmunoassay in 200 male controls yielded 11 (5.6 per cent) and the p-nitrophenylphosphate enzymatic test yielded 7 (3.5 per cent) falsely positive results. In 90 non-prostatic human cancer sera 85 (94.5 per cent) were correctly classified as negative by the radioimmunoassay for prostatic acid phosphatase versus 66 (73 per cent) as negative by the enzymatic method. These data are discussed in terms of the merits of a radioimmunochemical approach for the measurement of human serum prostatic acid phosphatase.

Topics: Acid Phosphatase; Humans; Male; Nitrophenols; Organophosphorus Compounds; Prostate; Prostatic Neoplasms; Radioimmunoassay

Topics: Acid Phosphatase; Alkaline Phosphatase; Blood Platelets; Electrophoresis, Disc; Erythrocytes; Female; Humans; Indicators and Reagents; Isoenzymes; Leukocytes; Male; Methods; Naphthols; Nitrophenols; Phenolphthaleins; Phosphates; Prostatic Hyperplasia; Prostatic Neoplasms; Tartrates

Topics: Acid Phosphatase; Blood Cell Count; Blood Coagulation Tests; Blood Platelets; Carcinoma; Clot Retraction; Depression, Chemical; Heparin; Humans; Male; Nitrophenols; Prostatic Neoplasms; Temperature; Thrombocytopenia

Topics: Acid Phosphatase; Blood Platelets; Female; Formaldehyde; Germany, East; Humans; Male; Methods; Nitrophenols; Phosphoric Acids; Prostate; Prostatic Neoplasms; Sex Factors