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1.
J Biomol Tech ; 33(3)2022 10 15.
Article in English | MEDLINE | ID: mdl-36910579

ABSTRACT

Background: Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) is a powerful method commonly used to study global protein-DNA interactions including both transcription factors and histone modifications. We have found that the choice of ChIP-Seq library preparation protocol plays an important role in overall ChIP-Seq data quality. However, very few studies have compared ChIP-Seq libraries prepared by different protocols using multiple targets and a broad range of input DNA levels. Results: In this study, we evaluated the performance of 4 ChIP-Seq library preparation protocols (New England Biolabs [NEB] NEBNext Ultra II, Roche KAPA HyperPrep, Diagenode MicroPlex, and Bioo [now PerkinElmer] NEXTflex) on 3 target proteins, chosen to represent the 3 typical signal enrichment patterns in ChIP-Seq experiments: sharp peaks (H3K4me3), broad domains (H3K27me3), and punctate peaks with a protein binding motif (CTCF). We also tested a broad range of different input DNA levels from 0.10 to 10 ng for H3K4me3 and H3K27me3 experiments. Conclusions: Our results suggest that the NEB protocol may be better for preparing H3K4me3 (and potentially other histone modifications with sharp peak enrichment) libraries; the Bioo protocol may be better for preparing H3K27me3 (and potentially other histone modifications with broad domain enrichment) libraries, and the Diagenode protocol may be better for preparing CTCF (and potentially other transcription factors with well-defined binding motifs) libraries.  For ChIP-Seq experiments using novel targets without a known signal enrichment pattern, the NEB protocol might be the best choice, as it performed well for each of the 3 targets we tested across a wide array of input DNA levels.


Subject(s)
Chromatin Immunoprecipitation Sequencing , Histones , Histones/metabolism , Gene Library , DNA , High-Throughput Nucleotide Sequencing/methods , Transcription Factors/metabolism , Sequence Analysis, DNA/methods
2.
Eur Rev Med Pharmacol Sci ; 25(1): 116-126, 2021 01.
Article in English | MEDLINE | ID: mdl-33506899

ABSTRACT

OBJECTIVE: Dysregulated lipid metabolism has been reported in the progression of hepatocellular carcinoma (HCC). In the present study, we investigated the molecular characteristics of lipid-metabolism-related genes (IMRGs) as prognostic markers for HCC. MATERIALS AND METHODS: Multi-dimensional bioinformatics analyses were performed to comprehensively analyze IMRGs, and to construct prognostic prediction signatures. RESULTS: Data of 770 HCC patients and their corresponding 776 IMRGs were downloaded from three databases. Patients were classified into 2 molecular clusters that were associated with overall survival, clinical characteristics, and immune cells. The biological functions of the IMRGs differentially expressed between the 2 clusters were associated with tumor-related metabolic pathways. A 6 IMRG signature (6-IS), consisting of FMO3, SLC11A1, RNF10, KCNH2, ME1, and ZIC2, was established as an independent prognostic factor for HCC. The performance of the signature of 6-IS prognostic was verified in a validation set and compared to an external data set. It was revealed that the 6-IS could effectively predict the prognosis of patients with HCC. CONCLUSIONS: This study provides new insights into the role of IMRGs in the pathogenesis of HCC, and presents a novel signature (6-IS) to predict the prognosis of HCC.


Subject(s)
Algorithms , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , Lipid Metabolism/genetics , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/diagnosis , Humans , Liver Neoplasms/diagnosis , Prognosis
3.
Oncogene ; 35(46): 5963-5976, 2016 11 17.
Article in English | MEDLINE | ID: mdl-26804168

ABSTRACT

Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.


Subject(s)
Drug Resistance, Neoplasm , Epithelium/metabolism , Epithelium/pathology , Forkhead Transcription Factors/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Androgens/metabolism , Animals , Benzamides , Cell Line, Tumor , Disease Models, Animal , Epithelial-Mesenchymal Transition/genetics , Forkhead Transcription Factors/genetics , Gene Expression , Humans , MAP Kinase Signaling System/drug effects , Male , Mice , Models, Biological , Neoplasm Grading , Nitriles , Phenotype , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostate-Specific Antigen/metabolism , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Recurrence , Xenograft Model Antitumor Assays , Zinc Finger E-box-Binding Homeobox 1/metabolism
4.
Oncogene ; 30(36): 3833-45, 2011 Sep 08.
Article in English | MEDLINE | ID: mdl-21499299

ABSTRACT

Cancer cell molecular mimicry of stem cells (SC) imbues neoplastic cells with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to show oncogenic potential. We have recently reported that short-hairpin RNA-mediated knockdown of the embryonic stem cell (ESC) self-renewal gene NANOG significantly reduced the clonogenic and tumorigenic capabilities of various cancer cells. In this study, we sought to test the potential pro-tumorigenic functions of NANOG, particularly, in prostate cancer (PCa). Using qRT-PCR, we first confirmed that PCa cells expressed NANOG mRNA primarily from the NANOGP8 locus on chromosome 15q14. We then constructed a lentiviral promoter reporter in which the -3.8-kb NANOGP8 genomic fragment was used to drive the expression of green fluorescence protein (GFP). We observed that NANOGP8-GFP(+) PCa cells showed cancer stem cell (CSC) characteristics such as enhanced clonal growth and tumor regenerative capacity. To further investigate the functions and mechanisms of NANOG in tumorigenesis, we established tetracycline-inducible NANOG-overexpressing cancer cell lines, including both PCa (Du145 and LNCaP) and breast (MCF-7) cancer cells. NANOG induction promoted drug resistance in MCF-7 cells, tumor regeneration in Du145 cells and, most importantly, castration-resistant tumor development in LNCaP cells. These pro-tumorigenic effects of NANOG were associated with key molecular changes, including an upregulation of molecules such as CXCR4, IGFBP5, CD133 and ALDH1. The present gain-of-function studies, coupled with our recent loss-of-function work, establish the integral role for NANOG in neoplastic processes and shed light on its mechanisms of action.


Subject(s)
Androgens/metabolism , Homeodomain Proteins/physiology , Neoplastic Stem Cells/cytology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Animals , Carcinoma, Embryonal/pathology , Cell Line, Tumor , Chromatin Immunoprecipitation , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Male , Mice , Mice, Inbred NOD , Mice, SCID , Models, Genetic , Nanog Homeobox Protein , Phenotype , RNA, Messenger/metabolism
5.
Oncogene ; 29(30): 4261-75, 2010 Jul 29.
Article in English | MEDLINE | ID: mdl-20514017

ABSTRACT

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate, and is often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we generated prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks arachidonic acid-metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67(+) cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem or progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia or carcinoma and, mechanistically, prostate lobes (especially those of 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-betagal, p27(Kip1) and heterochromatin protein 1gamma staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.


Subject(s)
Arachidonate 15-Lipoxygenase/physiology , Cellular Senescence , Prostate/enzymology , Prostatic Hyperplasia/etiology , Animals , Arachidonate 15-Lipoxygenase/genetics , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p27 , Gene Expression Profiling , Intracellular Signaling Peptides and Proteins/analysis , Ki-67 Antigen/analysis , Male , Mice , Mice, Transgenic , Prostate/pathology
6.
Oncogene ; 25(12): 1696-708, 2006 Mar 16.
Article in English | MEDLINE | ID: mdl-16449977

ABSTRACT

CD44 is a multifunctional protein involved in cell adhesion and signaling. The role of CD44 in prostate cancer (PCa) development and progression is controversial with studies showing both tumor-promoting and tumor-inhibiting effects. Most of these studies have used bulk-cultured PCa cells or PCa tissues to carry out correlative or overexpression experiments. The key experiment using prospectively purified cells has not been carried out. Here we use FACS to obtain homogeneous CD44(+) and CD44(-) tumor cell populations from multiple PCa cell cultures as well as four xenograft tumors to compare their in vitro and in vivo tumor-associated properties. Our results reveal that the CD44(+) PCa cells are more proliferative, clonogenic, tumorigenic, and metastatic than the isogenic CD44(-) PCa cells. Subsequent molecular studies demonstrate that the CD44(+) PCa cells possess certain intrinsic properties of progenitor cells. First, BrdU pulse-chase experiments reveal that CD44(+) cells colocalize with a population of intermediate label-retaining cells. Second, CD44(+) PCa cells express higher mRNA levels of several 'stemness' genes including Oct-3/4, Bmi, beta-catenin, and SMO. Third, CD44(+) PCa cells can generate CD44(-) cells in vitro and in vivo. Fourth, CD44(+) PCa cells, which are AR(-), can differentiate into AR(+) tumor cells. Finally, a very small percentage of CD44(+) PCa cells appear to undergo asymmetric cell division in clonal analyses. Altogether, our results suggest that the CD44(+) PCa cell population is enriched in tumorigenic and metastatic progenitor cells.


Subject(s)
Cell Line, Tumor/pathology , Hyaluronan Receptors/metabolism , Neoplasm Metastasis/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Stem Cells/physiology , Animals , Cell Line, Tumor/cytology , Cell Proliferation , Flow Cytometry , Humans , Male , Neoplasm Transplantation , Transplantation, Heterologous
7.
J Surg Res ; 109(1): 8-15, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12591229

ABSTRACT

BACKGROUND: Ischemia-reperfusion injury continues to represent a significant challenge to successful lung transplantation. Traditional pulmonary ischemic protection is performed using hypothermic hyperkalemic depolarizing solutions to reduce the metabolic demands of the ischemic organ. Measures to further reduce the effects of ischemic injury have focused on the reperfusion period. We tested the hypothesis that novel physiologic hyperpolarizing solutions-using ATP-dependent potassium channel (K(ATP)) openers-given at the induction of ischemia, will reduce cellular injury and provide superior graft function even after prolonged periods of ischemia. METHODS: An isolated blood-perfused ventilated rabbit lung model was used to study lung injury. Airway, left atrial, and pulmonary artery pressures were measured continuously during the 2-h reperfusion period. Oxygenation, as a surrogate of graft function, was measured using intermittent blood gas analysis of paired left atrial and pulmonary artery blood samples. Graft function was measured by oxygen challenge technique (F(i)O(2) = 1.0). Wet-to-dry ratio was measured at the conclusion of the 2-h reperfusion period. Control (Group I) lungs were perfused with modified Euro-Collins solution (depolarizing) and reperfused immediately (no ischemia). Traditional protection lungs were perfused with modified Euro-Collins flush solution and stored for 4 h (Group II) or 18 h (Group III) at 4 degrees C before reperfusion. Novel protection (Group IV) lungs were protected with a hyperpolarizing solution containing 100 nM Aprikalim, a specific K(ATP) channel opener, added to the modified Euro-Collins flush solution and underwent 18 h of ischemic storage at 4 degrees C before reperfusion. RESULTS: Profound graft failure was measured after 18 h of ischemic storage with traditional protection strategies (Group III). Graft function was preserved by protection with hyperpolarizing solutions even for prolonged ischemic periods (Group IV). Wet-to-dry weight ratio, airway, left atrial, and pulmonary artery pressures were not significantly different between the groups. CONCLUSIONS: We have created a model of predictable lung injury. Membrane hyperpolarization with a K(ATP) channel opener (PCO) provides superior prolonged protection from ischemia-reperfusion injury in an in vitro model of pulmonary transplantation.


Subject(s)
Lung Transplantation/methods , Picolines/administration & dosage , Pyrans/administration & dosage , Animals , Blood Pressure , Female , Heart Atria , Hypertonic Solutions , In Vitro Techniques , Ion Channel Gating/drug effects , Lung Transplantation/adverse effects , Male , Organ Preservation Solutions , Oxygen/administration & dosage , Oxygen/blood , Potassium Channels/drug effects , Potassium Channels/physiology , Pulmonary Artery , Rabbits , Reperfusion Injury/etiology , Reperfusion Injury/prevention & control , Solutions
8.
EMBO J ; 20(18): 5261-8, 2001 Sep 17.
Article in English | MEDLINE | ID: mdl-11566889

ABSTRACT

Both thyroid hormone (TH) and retinoic acid (RA) induce purified rat oligodendrocyte precursor cells in culture to stop division and differentiate. We show that these responses are blocked by the expression of a dominant-negative form of p53. Moreover, both TH and RA cause a transient, immediate early increase in the same 8 out of 13 mRNAs encoding intracellular cell cycle regulators and gene regulatory proteins, but only if protein synthesis is inhibited. Platelet-derived growth factor (PDGF) withdrawal also induces these cells to differentiate, but we show that the intracellular mechanisms involved are different from those involved in the hormone responses: the changes in cell cycle regulators differ, and the differentiation induced by PDGF withdrawal (or that which occurs spontaneously in the presence of PDGF) is not blocked by the dominant-negative p53. These results suggest that TH and RA activate the same intracellular pathway leading to oligodendrocyte differentiation, and that this pathway depends on a p53 family protein. Differentiation that occurs independently of TH and RA apparently involves a different pathway. It is likely that both pathways operate in vivo.


Subject(s)
Oligodendroglia/physiology , Tumor Suppressor Protein p53/physiology , Animals , Cell Cycle Proteins/biosynthesis , Cell Cycle Proteins/genetics , Cell Differentiation , Cells, Cultured , Cyclin D , Cyclins/biosynthesis , Cyclins/genetics , Cycloheximide/pharmacology , Cytoplasm/metabolism , Immediate-Early Proteins/biosynthesis , Immediate-Early Proteins/genetics , Mutation , Oligodendroglia/drug effects , Platelet-Derived Growth Factor/physiology , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Thyroid Hormones/pharmacology , Transcriptional Activation , Tretinoin/pharmacology , Tumor Suppressor Protein p53/genetics
9.
Science ; 291(5505): 868-71, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11157165

ABSTRACT

Most mammalian somatic cells are thought to have a limited proliferative capacity because they permanently stop dividing after a finite number of divisions in culture, a state termed replicative cell senescence. Here we show that most oligodendrocyte precursor cells purified from postnatal rat optic nerve can proliferate indefinitely in serum-free culture if prevented from differentiating; various cell cycle-inhibitory proteins increase, but the cells do not stop dividing. The cells maintain high telomerase activity and p53- and Rb-dependent cell cycle checkpoint responses, and serum or genotoxic drugs induce them to acquire a senescence-like phenotype. Our findings suggest that some normal rodent precursor cells have an unlimited proliferative capacity if cultured in conditions that avoid both differentiation and the activation of checkpoint responses that arrest the cell cycle.


Subject(s)
Cell Division , Cellular Senescence , Oligodendroglia/cytology , Stem Cells/cytology , Animals , Apoptosis , Cell Culture Techniques , Cell Cycle , Cell Differentiation , Cells, Cultured , Contact Inhibition , Culture Media, Serum-Free , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Mutation , Nucleic Acid Synthesis Inhibitors , Oligodendroglia/physiology , Optic Nerve/cytology , Proteins/metabolism , Rats , Retinoblastoma Protein/metabolism , Stem Cells/physiology , Telomerase , Tumor Suppressor Protein p14ARF , Tumor Suppressor Protein p53/metabolism , ras Proteins/metabolism
10.
Neuron ; 28(1): 81-90, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11086985

ABSTRACT

It was previously shown that newly formed oligodendrocytes depend on axons for their survival, but the nature of the axon-derived survival signal(s) remained unknown. We show here that neuregulin (NRG) supports the survival of purified oligodendrocytes and aged oligodendrocyte precursor cells (OPCs) but not of young OPCs. We demonstrate that axons promote the survival of purified oligodendrocytes and that this effect is inhibited if NRG is neutralized. In the developing rat optic nerve, we provide evidence that delivery of NRG decreases both normal oligodendrocyte death and the extra oligodendrocyte death induced by nerve transection, whereas neutralization of endogenous NRG increases the normal death. These results suggest that NRG is an axon-associated survival signal for developing oligodendrocytes.


Subject(s)
Axons/metabolism , Cell Survival/physiology , Neuregulin-1/metabolism , Oligodendroglia/metabolism , Optic Nerve/metabolism , Animals , Axotomy , COS Cells , Cell Count , Cell Survival/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Coculture Techniques , Dose-Response Relationship, Drug , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Neuregulin-1/genetics , Neuregulin-1/pharmacology , Oligodendroglia/cytology , Oligodendroglia/drug effects , Optic Nerve/cytology , Optic Nerve/drug effects , Optic Nerve/growth & development , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Stem Cells/cytology , Stem Cells/drug effects , Transfection
11.
J Cell Biol ; 148(5): 971-84, 2000 Mar 06.
Article in English | MEDLINE | ID: mdl-10704447

ABSTRACT

Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve and cultured them in serum-free medium containing platelet-derived growth factor (PDGF), the main mitogen for OPCs, but in the absence of thyroid hormone in order to inhibit their differentiation into oligodendrocytes. We find that many of the cells continue to proliferate for more than a year and progressively acquire a number of the characteristics of OPCs isolated from adult optic nerve. These findings suggest that OPCs have an intrinsic maturation program that progressively changes the cell's phenotype over many months. When we culture the postnatal OPCs in the same conditions but with the addition of basic fibroblast growth factor (bFGF), the cells acquire these mature characteristics much more slowly, suggesting that the combination of bFGF and PDGF, previously shown to inhibit OPC differentiation, also inhibits OPC maturation. The challenge now is to determine the molecular basis of such a protracted maturation program and how the program is restrained by bFGF.


Subject(s)
Cell Differentiation/physiology , Oligodendroglia/cytology , Oligodendroglia/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Animals , Animals, Newborn , Antigens, Differentiation/biosynthesis , Cell Count/drug effects , Cell Cycle/drug effects , Cell Death , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Size/drug effects , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Fibroblast Growth Factor 2/pharmacology , Humans , Oligodendroglia/drug effects , Optic Nerve/cytology , Phenotype , Platelet-Derived Growth Factor/pharmacology , Rats , Rats, Sprague-Dawley , Stem Cells/drug effects , Time
12.
Ann Surg ; 230(3): 404-11; discussion 411-3, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10493487

ABSTRACT

OBJECTIVE: To examine the association of surgeon and hospital case volumes with the short-term outcomes of in-hospital death, total hospital charges, and length of stay for resection of colorectal carcinoma. METHODS: The study design was a cross-sectional analysis of all adult patients who underwent resection for colorectal cancer using Maryland state discharge data from 1992 to 1996. Cases were divided into three groups based on annual surgeon case volume--low (< or =5), medium (5 to 10), and high (>10)--and hospital volume--low (<40), medium (40 to 70), and high (> or =70). Poisson and multiple linear regression analyses were used to identify differences in outcomes among volume groups while adjusting for variations in type of resections performed, cancer stage, patient comorbidities, urgency of admission, and patient demographic variables. RESULTS: During the 5-year period, 9739 resections were performed by 812 surgeons at 50 hospitals. The majority of surgeons (81%) and hospitals (58%) were in the low-volume group. The low-volume surgeons operated on 3461 of the 9739 total patients (36%) at an average rate of 1.8 cases per year. Higher surgeon volume was associated with significant improvement in all three outcomes (in-hospital death, length of stay, and cost). Medium-volume surgeons achieved results equivalent to high-volume surgeons when they operated in high- or medium-volume hospitals. CONCLUSIONS: A skewed distribution of case volumes by surgeon was found in this study of patients who underwent resection for large bowel cancer in Maryland. The majority of these surgeons performed very few operations for colorectal cancer per year, whereas a minority performed >10 cases per year. Medium-volume surgeons achieved excellent outcomes similar to high-volume surgeons when operating in medium-volume or high-volume hospitals, but not in low-volume hospitals. The results of low-volume surgeons improved with increasing hospital volume but never equaled those of the high-volume surgeons.


Subject(s)
Colonic Neoplasms/mortality , Colonic Neoplasms/surgery , Digestive System Surgical Procedures/statistics & numerical data , General Surgery/statistics & numerical data , Hospitals/statistics & numerical data , Rectal Neoplasms/mortality , Rectal Neoplasms/surgery , Aged , Clinical Competence , Cross-Sectional Studies , Diagnosis-Related Groups , Female , Humans , Male , Regression Analysis
13.
Cancer Res ; 59(17): 4343-55, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10485482

ABSTRACT

We recently developed a class of novel anti-prostate cancer compounds, cyclic hydroxamates that elicit a potent apoptotic response in many tumor cells cultured in vitro (D.G. Tang et al., Biochem. Biophys. Res. Commun., 242: 380-384, 1998). The lead compound, termed BMD188, induces programmed cell death in a variety of prostate cancer cells in vitro as well as in vivo (L. Li et al., Anticancer Res., 19: 51-70, 1999). BMD188 kills androgen-independent prostate cancer cells as well as prostate cancer cells with a multidrug-resistance phenotype. The apoptotic effect of BMD188 in prostate cancer cells does not depend on cell cycle, p53 status, or its purported target, arachidonate 12-lipoxygenase, but does require caspase activation and seems to involve mitochondria. To synthesize more specific and effective anti-prostate cancer hydroxamic acid compounds, it is important to understand their mechanism(s) of action. In the present study, we studied the role of mitochondrial respiratory chain (MRC) in BMD188-induced apoptosis in androgen-independent prostate cancer PC3 cells and compared its effect with that of staurosporine (STS), a widely used apoptosis inducer. Several lines of evidence indicate that BMD188-induced cell death depends on MRC: (a) the death could be significantly inhibited by several complex-specific respiration inhibitors; (b) respiration-deficient rho0 cells were more resistant than wild-type parent cells to apoptosis induction by BMD188; and (c) BMD188 induced a rapid increase in reactive oxygen species in mitochondria, an up-regulation of cytochrome c oxidase subunits, a biphasic alteration (i.e., an early hyperpolarization, followed by later hypopolarization) in the mitochondrial membrane potential (delta psi(m)), dramatic changes in mitochondrial morphology and distribution prior to caspase activation, and an abnormal proliferation of mitochondria at the ultrastructural level. By contrast, STS-induced PC3 apoptosis seemed not to depend on MRC. Taken together, the data suggest that the MRC represents a functional target for anti-prostate cancer hydroxamates.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Electron Transport/drug effects , Hydroxamic Acids/pharmacology , Mitochondria/metabolism , Piperidones/pharmacology , Prostatic Neoplasms/drug therapy , Caspase 3 , Caspases/physiology , Humans , Male , Membrane Potentials/drug effects , Mitochondria/drug effects , Prostaglandin-Endoperoxide Synthases/biosynthesis , Reactive Oxygen Species , Staurosporine/pharmacology , Tumor Cells, Cultured
14.
Anticancer Res ; 19(1A): 61-9, 1999.
Article in English | MEDLINE | ID: mdl-10226525

ABSTRACT

BACKGROUND: In the preceding paper, we demonstrated that, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], a newly synthesized cyclic hydroxamic acid compound, induces potent apoptotic death of prostate cancer cells in vitro. In this project, we studied the in vivo pharmacokinetic behavior and anti-tumor efficacy of this novel compound. MATERIALS AND METHODS: A bioavailability/elimination study was first performed using radiolabeled BMD188 administered to rats through intraperitoneal (i.p.), intravenous (i.v). or oral (p.o.) routes. Based on these pharmacokinetic data as well as pilot experiments on in vivo toxicity, two sets of efficacy studies, with i.p. administered BMD188, were performed in SCID mice or athymic nude mice which had been orthotopically transplanted with Du145 human prostate cancer cells. Tumor growth rate was measured and the final tumor weights and sizes determined. Subsequently, histopathological data were obtained and tumor tissue sections were used for apoptosis (i.e., TUNEL) staining. RESULTS: The pharmacokinetic studies revealed low (approximately 8%) absorption through the p.o. route and high (approximately 70%) absorption through the i.p. route. The average plasma half life (T1/2) of BMD188 was approximately 50 h. Post-absorption, plasma elimination of radioactivity was similar to that in animals given [3H]-188 intravenously. The in vivo efficacy results indicate that i.p. administered BMD188 significantly inhibited the primary growth and local invasion of Du145 prostate cancer cells orthotopically implanted into SCID or athymic nude mice. The tumor-inhibitory effect of BMD188 was due to apoptosis induction in vivo, as revealed by histological analysis as well as TUNEL staining of the tumor tissue sections. CONCLUSION: Collectively, the preceding in vitro and the current in vivo studies suggest that BMD188 and its analogs may find clinical applications in the treatment of prostate cancer patients by inducing apoptotic death of prostate cancer cells.


Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Hydroxamic Acids/therapeutic use , Piperidones/therapeutic use , Prostatic Neoplasms/drug therapy , Animals , Humans , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , Male , Mice , Mice, Inbred CBA , Mice, Nude , Mice, SCID , Neoplasm Transplantation , Piperidones/pharmacokinetics , Piperidones/pharmacology , Rats , Rats, Sprague-Dawley , Tumor Cells, Cultured
15.
Anticancer Res ; 19(1A): 51-60, 1999.
Article in English | MEDLINE | ID: mdl-10226524

ABSTRACT

BACKGROUND: Prostate cancer is the most frequently diagnosed malignancy in the Western countries. Apoptosis-targeted drug development could represent a specific and effective weapon against the disease (Tang and Porter, 32: 284-293, 1997). We previously demonstrated that the arachidonate 12-lipoxygenase and its metabolic products could function as survival factors for many solid tumors (Tang et al., Proc. Natl. Acad. Sci. USA 93: 5241-5246, 1996; Tang and Honn, J. Cell. Physiol. 172: 155-170, 1997). MATERIALS AND METHODS: In this study, we synthesized a series of novel cyclic hydroxamic acid compounds that demonstrated varying degrees of inhibitory effects on the arachidonate 12-lipoxygenase. Subsequently we studied the effects of these novel compounds on human prostate cancer cells. First, all these compounds were screened on androgen-independent PC3 adenocarcinoma cells. Second, based on the results (i.e., the LD50 values) of the primary, secondary and tertiary screening, lead compounds were determined. Third, the lead compounds were utilized to study their cytotoxic effects on various prostate cancer cells as well as several types of normal cells. Finally, the molecular nature of the cell death was thoroughly characterized and the potential mechanisms of cell death were determined. RESULTS: About 30% of the compounds screened induced a strong apoptotic death of androgen-independent prostate cancer cells, PC3, with an LD50 mostly at 10-20 microM. A lead compound, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], was subsequently identified which inhibited the growth of PC3 cells with an LD50 at approximately 10 microM. Comparative studies indicated that BMD188 induced a more potent apoptotic response in PC3 cells than several conventional chemotherapeutic drugs. Furthermore, unlike the above drugs, BMD188 could induce 100% apoptosis in tumor cells. BMD188 also caused apoptosis of other types of prostate cancer cells including cells with multidrug resistance phenotype, independent of the androgen-dependence and p53 status. By contrast, BMD188 generally demonstrated 2-5 fold lower cytotoxicity towards several normal cell types including normal prostate epithelial cells. The growth inhibition by BMD188 was due to apoptosis induction as evidenced by DNA ladder formation, PARP [poly(ADP-ribose)polymerase] cleavage, and typical apoptotic morphology. BMD188-induced apoptosis does not depend on its inhibitory effects on lipoxygenase since target cells (i.e., PC3 and Du145) did not express the lipoxygenase mRNA and protein. In contrast, the apoptosis-inducing effect of BMD188 in PC3 cells could be significantly inhibited by serine protease inhibitors TPCK and TLCK as well as by caspase inhibitors DEVD and zVAD. The involvement of caspases in the apoptotic effects of BMD188 was further confirmed by the activation of caspase-3 (CPP32). In the accompanying paper, we show that BMD188 also inhibits the primary growth and local invasion of Du145 prostate cancer cells orthotopically implanted into the SCID or athymic nude mice. CONCLUSION: The data presented here suggest that these novel cyclic hydroxamic acid compounds, via induction of apoptotic death, may find potential clinical applications in the treatment of human prostate cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Hydroxamic Acids/pharmacology , Piperidones/pharmacology , Prostatic Neoplasms/drug therapy , Animals , Caspase 3 , Caspases/metabolism , DNA Fragmentation/drug effects , Humans , Male , Mice , Poly(ADP-ribose) Polymerases/metabolism , Prostatic Neoplasms/pathology , Serine Endopeptidases/metabolism
16.
Anticancer Res ; 19(1A): 403-8, 1999.
Article in English | MEDLINE | ID: mdl-10226574

ABSTRACT

BACKGROUND: Apoptosis, or programmed cell death, is characterized by certain distinct morphological and biochemical features. Most chemotherapeutic drugs exert their anti-tumor effects by inducing apoptosis. Therefore, an effective compound inducing apoptosis appears to be a relevant strategy to suppress various human tumors. In a search for tumor inhibitors from various kinds of plants, we found that extracts from Solanum muricatum (CSG) can inhibit tumor growth both in vivo and in vitro by inducing apoptosis. MATERIALS AND METHODS: A lyophilized aqueous fraction extracted from Solanum muricatum (CSG4) was used in this study. The human cell lines tested include: prostate (PC3, DU145), stomach (MKN45), liver (QGY-7721, SK-HEP-1), breast (MDA-MB-435), ovarian (OVCAR), colon (HT29) and lung (NCI-H209) cancer cells; NHP (prostate), HUVEC (umbilical vein endothelial cell), and WI-38 (lung diploid fibroblasts) normal cells. The cell survival was determined by either Cell Titer MTS cell proliferation kit or trypan blue dye exclusion assay. The apoptosis was analyzed by (a) apoptotic morphology by light microscopy; (b) DNA ladder formation; (c) PARP cleavage assay. RESULTS: a) CSG possesses selective cytotoxic activity against all the tumor cell lines being tested. The LD50 value is 561-825 micrograms/ml. b) CSG showed a much lower cytotoxicity to NHP, HUVEC and WI-38 normal cell lines with LD50 value being 2.8-3.2 mg/ml, which is 3-6 fold higher than on tumor cells. c) The in vivo study demonstrated that injection of CSG (100 micrograms) directly into tumor mass can reduce the tumor volume dramatically in nude mice inoculated with MKN45 gastric cancer cells. d) CSG-mediated tumor growth inhibition is through induction of apoptotic cell death, as manifested by (a) typical apoptotic morphology; (b) DNA ladder formation; and (c) PARP cleavage assay. CONCLUSION: Taken together, the present study suggests, for the first time, that CSG may represent promising new chemical entity which preferentially targets various tumor cells by triggering apoptosis.


Subject(s)
Anticarcinogenic Agents/pharmacology , Apoptosis/drug effects , Plants, Medicinal , Animals , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Male , Mice , Mice, Nude , Plant Extracts/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Stomach Neoplasms/drug therapy , Stomach Neoplasms/pathology , Tumor Cells, Cultured
17.
Prostate ; 38(2): 118-25, 1999 Feb 01.
Article in English | MEDLINE | ID: mdl-9973097

ABSTRACT

BACKGROUND: PSP94 (prostate secretory protein of 94 aa; also called PIP), one of the predominant proteins secreted into the seminal fluid, was proposed as an independent diagnostic/prognostic marker for prostate cancers. It was also shown to inhibit rat prostate cancer growth. In this study, we investigated the effect of purified PSP94 on the growth of androgen-independent human prostate cancer cells (PC3) and its potential mechanism of action. METHODS AND RESULTS: PSP94, in a dose- and time-dependent manner, inhibited the growth of PC3 cells. The protein demonstrated a stronger inhibitory effect on the colony-forming ability of PC3 cells in soft agar. A daily injection of PSP94 at 5 microg/kg/body weight resulted in a 50-60% inhibition in the growth of PC3 xenografts in athymic mice. PC3 cell growth inhibition by PSP94 resulted from cell death characteristic of morphological apoptosis, which was confirmed by dual fluorescence microscopy, electron microscopy, and DNA fragmentation assays. Mechanistic studies indicated that PSP94 enhanced the expression of proapoptotic protein Bax without affecting Bcl-2 levels. CONCLUSIONS: This study suggests that PSP94 may represent a novel, apoptosis-based, antitumor agent applicable to the treatment of hormone-refractory human prostate cancers.


Subject(s)
Apoptosis , Peptides/physiology , Prostatic Neoplasms/pathology , Prostatic Secretory Proteins , Androgens , Animals , Apoptosis/drug effects , Biomarkers, Tumor , Blotting, Western , Clone Cells/drug effects , Dose-Response Relationship, Drug , Humans , Male , Mice , Mice, Nude , Microscopy, Fluorescence , Neoplasms, Hormone-Dependent/pathology , Peptides/administration & dosage , Peptides/pharmacology , Rats , Time Factors , Transplantation, Heterologous , Tumor Cells, Cultured
18.
J Urol ; 161(3): 970-6, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10022736

ABSTRACT

PURPOSE: Understanding growth regulation in hormone-refractory prostate cancer may provide avenues for novel treatment interventions. This study was conducted to characterize the expression of the receptor (FSHR) for follicle-stimulating hormone (FSH) in androgen-independent prostate cancer cell lines and in human malignant prostate tissues. MATERIALS AND METHODS: Western blotting, immunohistochemistry (IHC), and flow cytometric analysis were used to study the expression of FSHR. The effect of FSH on cell growth and clonogenicity was studied using proliferation and clonogenic assays. RESULTS: Immunohistochemistry revealed expression of FSH in PC3 and Du145 cells. FSHR was identified in PC3 and Du145 cells, as well as in human adenocarcinoma of the prostate. The specificity of the FSHR detected on prostate cancer tissues or cells by IHC and Western blotting was confirmed by preabsorbing the antibodies with the immunizing antigens. Stimulation of these hormone-refractory cells with FSH triggered a proliferative response in vitro, suggesting that the receptor is biologically active. CONCLUSION: Hormone-refractory prostate cancer cells express FSH and biologically active FSHR. Our results suggest that FSHR and its ligand may play a role in the regulation of the growth of hormone-refractory prostate cancers.


Subject(s)
Prostatic Neoplasms/metabolism , Receptors, FSH/biosynthesis , Androgen Antagonists/therapeutic use , Humans , Male , Prostatic Neoplasms/drug therapy , Treatment Failure , Tumor Cells, Cultured
19.
Pathol Oncol Res ; 4(3): 179-90, 1998.
Article in English | MEDLINE | ID: mdl-9761936

ABSTRACT

A newly synthesized cyclic hydroxamic acid compound, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], was found to induce the apoptotic death of cultured prostate cancer cells by activating caspase-3. Orally administered BMD188 significantly inhibited the primary growth of prostate cancer cells (Du145) orthotopically implanted into SCID mice. Mechanistic studies indicated that BMD188 did not alter the protein levels of several Bcl-2 family members. In contrast, the BMD188 effect required three essential factors: reactive oxygen species (ROS), the mitochondrial respiratory chain function, and proteases. First, the apoptosis-inducing effect of BMD188 could be blocked by ROS scavengers such as Desferal. Second, both BMD188-induced PARP cleavage as well as PC3 cell apoptosis could be dramatically inhibited by several complex-specific mitochondrial respiration blockers. The involvement of mitochondria was also supported by the observations that BMD188 dramatically altered the mitochondrial distribution and morphology without affecting the cellular ATP levels. Finally, the apoptosis-inducing effect of BMD188 in PC3 cells could be significantly inhibited by serine protease inhibitors (TPCK and TLCK) as well as by caspase inhibitors (zVAD-fmk and DEVD-CHO). Collectively, the present study suggests that BMD188 and its analogs may find clinical applications in the treatment of prostate cancer patients by inducing apoptotic death of prostate cancer cells.


Subject(s)
Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Caspases/metabolism , Endopeptidases/metabolism , Hydroxamic Acids/therapeutic use , Lipoxygenase Inhibitors/therapeutic use , Neoplasm Proteins/metabolism , Piperidones/therapeutic use , Prostatic Neoplasms/drug therapy , Animals , Antineoplastic Agents/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Caspase 3 , Enzyme Activation/drug effects , Humans , Hydroxamic Acids/antagonists & inhibitors , Hydroxamic Acids/pharmacology , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, SCID , Mitochondria/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Transplantation , Piperidones/antagonists & inhibitors , Piperidones/pharmacology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Reactive Oxygen Species , Serine Proteinase Inhibitors/pharmacology
20.
Cancer Res ; 58(15): 3466-79, 1998 Aug 01.
Article in English | MEDLINE | ID: mdl-9699682

ABSTRACT

This project was undertaken to study the survival properties of various prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells (LNCaP, PC3, and Du145), in the absence of trophic factors. Cell proliferation and cell death were quantitated by enumerating the number of live cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole dihydrochloride nuclear staining. These cells demonstrated an overall survivability in the order of BPH < NHP < LNCaP < PC3 < PCA < Du145. Upon growth factor deprivation, NHP/BPH cells rapidly underwent apoptosis, leading to a decreased number of live cells. PCA/PC3/Du145 cells, in contrast, demonstrated an initial phase of aggressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell proliferation, followed by a later phase in which apoptosis exceeded the cell proliferation. LNCaP cells demonstrated survival characteristics between those of NHP/BPH and PCA/PC3/Du145 cells. We concluded that the increased survivability in prostate cancer cells results from enhanced cell proliferation as well as decreased apoptosis. The molecular mechanisms for evasion of apoptosis in prostate cancer cells were subsequently investigated. Quantitative Western blotting was used to examine the protein expression of P53 and P21WAF-1, Bcl-2 and Bcl-X(L) (anti-apoptotic proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and down-regulated the expression of P21. Furthermore, NHP and BPH cells endogenously expressed little or no Bcl-2. In sharp contrast, prostate cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 and/or BclX(L), lost response to withdrawal-induced up-regulation of Bax/Bad/Bak or decreased or even completely lost Bax expression and expressed some novel proteins such as P25 and P54/55 complex. These data together suggest that prostate cancer cells may use multiple molecular mechanisms to evade apoptosis, which, together with increased proliferation, contribute to extended survivability of prostate cancer cells in the absence trophic factors.


Subject(s)
Apoptosis/physiology , Neoplasm Proteins/physiology , Prostatic Neoplasms/pathology , Carrier Proteins/physiology , Cell Division/physiology , Cell Survival/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/physiology , Humans , Male , Membrane Proteins/physiology , Prostate/cytology , Prostate/metabolism , Prostatic Hyperplasia/metabolism , Prostatic Hyperplasia/pathology , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , Tumor Suppressor Protein p53/physiology , bcl-2 Homologous Antagonist-Killer Protein , bcl-2-Associated X Protein , bcl-Associated Death Protein , bcl-X Protein
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