bluuebunny/crossref_metadata_2025 huggingface dataset:
Gain of OGP, an Estrogen-Regulated Oviduct-Specific Glycoprotein, Is Associated with the Development of Endometrial Hyperplasia and Endometrial Cancer
Authors: Woo Michelle M. M. , Alkushi Abdulmohsen , Verhage Harold G. , Magliocco Anthony M. , Leung Peter C. K. , Gilks C. Blake , Auersperg Nelly
Published: September 2005

Purpose: Lesions in the endometrium are difficult to differentially diagnose. The present study examined whether oviduct-specific glycoprotein is differentially expressed in normal, hyperplastic, and malignant endometrium.

Experimental Design: The expression of oviduct-specific glycoprotein was characterized by immunohistochemical methods with whole sections of endometrium from 90 women. An endometrial cancer tissue microarray with 200 cases of endometrial cancer was also assessed for oviduct-specific glycoprotein, estrogen receptor, and PTEN expression.

Results: In normal endometrium, there was focal oviduct-specific glycoprotein expression in the basalis layer, where the stem cells reside, in 10 of 15 cases. On average, atypical hyperplastic endometria stained more intensely than hyperplastic endometria (P = 0.017), whereas the percentage of positively stained cells was not significantly different. The mean staining indices (intensity × percentage of positive cells score) for hyperplasia and atypical hyperplastic were 4.7 and 5.5 and were significantly higher than staining indices seen in normal cycling endometria or well-differentiated endometrioid endometrial carcinomas (P < 0.0001 and P < 0.001, respectively). The endometrial cancer tissue microarray showed that of 139 endometrioid endometrial carcinomas, 11 cases were strongly oviduct-specific glycoprotein positive, whereas the other 128 cases were negative or weakly positive. Analysis of Kaplan-Meier curves with log-rank statistics showed a trend toward significance, with strong oviduct-specific glycoprotein staining serving as a predictor of good prognosis (P = 0.1). There was a significant positive correlation between oviduct-specific glycoprotein staining and loss of PTEN in the cases of endometrial cancer (P = 0.004).

Conclusions: Oviduct-specific glycoprotein may be a useful diagnostic adjunct to more accurately classify premalignant and early malignant change in the endometrium, improving on the current irreproducible histologic classification system.
Transcription Factor AP-2γ Is a Developmentally Regulated Marker of Testicular CarcinomaIn situand Germ Cell Tumors
Authors: Hoei-Hansen Christina E. , Nielsen John E. , Almstrup Kristian , Sonne Si Brask , Graem Niels , Skakkebaek Niels E. , Leffers Henrik , Meyts Ewa Rajpert-De
Published: September 2005

Purpose: Transcription factor activator protein-2γ (TFAP2C, AP-2γ) was reported previously in extraembryonic ectoderm and breast carcinomas but not in the testis. In our recent gene expression study we detected AP-2γ in carcinoma in situ testis (CIS, or intratubular germ cell neoplasia), precursor of testicular germ cell tumors. In this study we aimed to investigate the expression pattern of AP-2γ and to shed light on this factor in germ cell differentiation and the pathogenesis of germ cell neoplasia.

Experimental Design: We analyzed expression pattern of AP-2γ at the RNA and protein level in normal human tissues and a panel of tumors and tumor-derived cell lines. In the gonads, we established the ontogeny of expression of AP-2γ in normal and dysgenetic samples. We also investigated the regulation of AP-2γ by steroids and retinoic acid.

Results: We detected abundant AP-2γ in testicular CIS and in testicular germ cell tumors of young adults and confirmed differential expression of AP-2γ in somatic tumors. We found that AP-2γ expression was regulated by retinoic acid in an embryonal carcinoma cell line (NT2). The investigation of ontogeny of AP-2γ protein expression in fetal gonads revealed that it was confined to oogonia/gonocytes and was down-regulated with germ cell differentiation. In some prepubertal intersex cases, AP-2γ was detected outside of the normal window of expression, probably marking neoplastic transformation of germ cells.

Conclusions: AP-2γ is developmentally regulated and associated with the undifferentiated phenotype in germ cells. This transcription factor may be involved in self-renewal and survival of immature germ cells and tissue-specific stem cells. AP-2γ is a novel marker of testicular CIS and CIS-derived tumors.
Factors Affecting Cytochrome P-450 3A Activity in Cancer Patients
Authors: Baker Sharyn D. , van Schaik Ron H. N. , Rivory Laurent P. , ten Tije Albert J. , Dinh Kimberly , Graveland Wilfried J. , Schenk Paul W. , Charles Kellie A. , Clarke Stephen J. , Carducci Michael A. , McGuire William P. , Dawkins Fitzroy , Gelderblom Hans , Verweij Jaap , Sparreboom Alex
Published: September 2005

Purpose: The purpose is to identify the demographic, physiologic, and inheritable factors that influence CYP3A activity in cancer patients

Experimental Design: A total of 134 patients (62 females; age range, 26 to 83 years) underwent the erythromycin breath test as a phenotyping probe of CYP3A. Genomic DNA was screened for six variants of suspected functional relevance in CYP3A4 (CYP3A4*1B, CYP3A4*6, CYP3A4*17, and CYP3A4*18) and CYP3A5 (CYP3A5*3C and CYP3A5*6).

Results: CYP3A activity (AUC0–40min) varied up to 14-fold in this population. No variants in the CYP3A4 and CYP3A5 genes were a significant predictor of CYP3A activity (P > 0.2954). CYP3A activity was reduced by ∼50% in patients with concurrent elevations in liver transaminases and alkaline phosphatase or elevated total bilirubin (P < 0.001). In a multivariate analysis, CYP3A activity was not significantly influenced by age, sex, and body size measures (P > 0.05), but liver function combined with the concentration of the acute-phase reactant, α-1 acid glycoprotein, explained ∼18% of overall variation in CYP3A activity (P < 0.001).

Conclusions: These data suggest that baseline demographic, physiologic, and chosen genetic polymorphisms have a minor impact on phenotypic CYP3A activity in patients with cancer. Consideration of additional factors, including the inflammation marker C-reactive protein, as well as concomitant use of other drugs, food constituents, and complementary and alternative medicine with inhibitory and inducible effects on CYP3A, is needed to reduce variation in CYP3A and treatment outcome to anticancer therapy.
Paclitaxel-Loaded Gelatin Nanoparticles for Intravesical Bladder Cancer Therapy
Authors: Lu Ze , Yeh Teng-Kuang , Tsai Max , Au Jessie L.-S. , Wientjes M. Guill
Published: September 2005

Purpose: The present report describes the development of paclitaxel-loaded gelatin nanoparticles for use in intravesical therapy of superficial bladder cancer. The commercial formulation of paclitaxel contains Cremophor, which forms micelles and thereby entraps the drug and reduces its partition across the urothelium.

Experimental Design: Paclitaxel-loaded gelatin nanoparticles were prepared using the desolvation method, and their physicochemical and biological properties were characterized.

Results: The size of the particles ranged from 600 to 1,000 nm and increased with the molecular weight of the gelatin polymer. Under optimal conditions, the yield was >80%, and the drug loading was 0.7%. Wide-angle X-ray diffraction analysis showed that the entrapped paclitaxel was present in an amorphous state, which has higher water solubility compared with the crystalline state. Identical, rapid drug release from nanoparticles was observed in PBS and urine, with ∼90% released at 37°C after 2 hours. Treatment with a protease (i.e., Pronase) rapidly degraded the nanoparticles, with half-lives of 23.8 minutes, 0.6 minute, and 0.4 minute in the presence of 0.01, 0.05, and 0.25 mg/mL Pronase, respectively. The paclitaxel-loaded nanoparticles were active against human RT4 bladder transitional cancer cells; the IC50 paclitaxel-equivalent concentrations were nearly identical to those of aqueous solutions of paclitaxel, i.e., ∼30 nmol/L (equivalent to ∼25 ng/mL) for 2-hour treatments and ∼4 nmol/L for 96-hour treatments. In dogs given an intravesical dose of paclitaxel-loaded particles, the drug concentrations in the urothelium and lamina propria tissue layers, where Ta and T1 tumors would be located, were 7.4 ± 4.3 μg/g (mean ± SD; 3 dogs; 9 tissue sections), which were 2.6× the concentrations we reported for dogs treated with the Cremophor formulation.

Conclusions: Paclitaxel-loaded gelatin nanoparticles represent a rapid release, biologically active paclitaxel formulation that can be used for intravesical bladder cancer therapy.
3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitor, Fluvastatin, as a Novel Agent for Prophylaxis of Renal Cancer Metastasis
Authors: Horiguchi Akio , Sumitomo Makoto , Asakuma Junichi , Asano Takako , Asano Tomohiko , Hayakawa Masamichi
Published: September 2005

Purpose: Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also called statins, are currently used widely as a safe, effective therapeutic in the treatment of hypercholesterolemia. Recently, statins have been recognized for their activity against cancer. In the present study, we examined the effect of a synthetic statin, fluvastatin, on the development of renal cancer.

Experimental Design: The effects of fluvastatin on cell viability, cell cycle, in vitro angiogenesis, and invasive properties were examined in murine renal cancer cell Renca. The changes in cell cycle-associated proteins, p21Waf1/Cip1 and p53, and rac1 phosphorylation were analyzed by Western blotting. The prophylactic efficacy of fluvastatin to murine pulmonary metastasis of Renca was examined.

Results: Fluvastatin inhibited in vitro growth of Renca cells in a time- and dose-dependent manner, with up to 70% inhibition at a concentration of 10 μmol/L. This inhibitory effect was due to cell cycle arrest at the G1 phase and induction of apoptosis accompanied by up-regulation of p21Waf1/Cip1 and p53. The invasive properties of Renca cells through Matrigel were inhibited by fluvastatin, with decreased phosphorylation of rac1. In vitro angiogenesis was also inhibited by fluvastatin. Furthermore, oral administration at doses of 1 to 10 mg/kg/d, for 12 days after inoculation of Renca cells via the tail vein, significantly decreased the amount of pulmonary metastasis.

Conclusions: Because our results suggest that fluvastatin may effectively inhibit in vitro tumor growth, invasion, angiogenesis, and metastasis of Renca cells, oral administration of fluvastatin could be a novel, safe, and effective agent for preventing metastasis of renal cancer.
In vitro and in vivo irinotecan-induced changes in expression profiles of cell cycle and apoptosis-associated genes in acute myeloid leukemia cells
Authors: Minderman Hans , Conroy Jeffrey M. , O'Loughlin Kieran L. , McQuaid Devin , Quinn Paul , Li Song , Pendyala Lakshmi , Nowak Norma J. , Baer Maria R.
Published: September 2005

Objective: To study irinotecan (CPT-11)–induced changes in expression profiles of genes associated with cell cycle control and apoptosis in myeloid leukemia cells in vitro and in vivo. Methods: HL60 cells were exposed to clinically achievable concentrations of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of CPT-11, and blood sampled from patients with acute myeloid leukemia and chronic myeloid leukemia in myeloid blast transformation treated with CPT-11. Gene expression changes were studied by cDNA microarray and correlated with biological responses by studying DNA distributions by flow cytometry. Results: cDNA microarray analysis showed down-regulation and up-regulation of specific cell cycle–associated genes, consistent with loss of S-phase cells and temporary delay of G1-S-phase transition seen by flow cytometry. Flow cytometry showed that cells in S phase during SN-38 exposure underwent apoptosis, whereas cells in G2-M and G1 were delayed in G1 and entered S phase only 6 to 8 hours after drug removal, consistent with the observed changes in gene expression. Proapoptotic changes in gene transcription included down-regulation of antiapoptotic genes and up-regulation of proapoptotic genes. Many gene expression changes observed following in vitro SN-38 exposure were also seen following in vivo administration of 10 or 15 mg/m2 CPT-11; notably, proapoptotic changes included reduced transcription of survivin pathway-associated genes and increased transcription of death receptor 5. Conclusion: CPT-11-induced changes in gene expression profiles in vitro and in vivo are consistent with temporary delay in G1-S transition and enhanced responsiveness to apoptosis, both of which may contribute to the synergistic interactions of this drug with antimetabolites.
Resveratrol-induced apoptotic death in human U251 glioma cells
Authors: Jiang Hao , Zhang Lijie , Kuo Jarret , Kuo Kelly , Gautam Subhash C. , Groc Laurent , Rodriguez Alba I. , Koubi David , Jackson Hunter Tangella , Corcoran George B. , Seidman Michael D. , Levine Robert A.
Published: September 2005

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Resveratrol has antiinflammatory and antioxidant properties, and also has potent anticancer properties. Human glioma U251 cells were used to understand the molecular mechanisms by which resveratrol acts as an anticancer agent, since glioma is a particularly difficult cancer to treat and eradicate. Our data show that resveratrol induces dose- and time-dependent death of U251 cells, as measured by lactate dehydrogenase release and internucleosomal DNA fragmentation assays. Resveratrol induces activation of caspase-3 and increases the cleavage of the downstream caspase substrate, poly(ADP-ribose) polymerase. Resveratrol-induced DNA fragmentation can be completely blocked by either a general caspase inhibitor (Z-VAD-FMK) or a selective caspase-3 inhibitor (Z-DEVD-FMK), but not by a selective caspase-1 inhibitor. Resveratrol induces cytochrome c release from mitochondria to the cytoplasm and activation of caspase-9. Resveratrol also increases expression of proapoptotic Bax and its translocation to the mitochondria. Resveratrol inhibits U251 proliferation, as measured by MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and induces G0/G1 growth arrest, as determined by flow cytometry. The cyclin-dependent kinase inhibitor, olomoucine, prevents cell cycle progression and resveratrol-induced apoptosis. These results suggest that multiple signaling pathways may underlie the apoptotic death of U251 glioma induced by resveratrol, which warrants further exploration as an anticancer agent in human glioma.
Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiation
Authors: Ayene Iraimoudi S. , Ford Lance P. , Koch Cameron J.
Published: September 2005

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiation-responsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to γ radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to γ radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments.
RNA expression profiling of normal and tumor cells following photodynamic therapy with 5-aminolevulinic acid–induced protoporphyrin IX in vitro
Authors: Wild Peter J. , Krieg Rene C. , Seidl Juergen , Stoehr Robert , Reher Kerstin , Hofmann Claudia , Louhelainen Jari , Rosenthal André , Hartmann Arndt , Pilarsky Christian , Bosserhoff Anja K. , Knuechel Ruth
Published: September 2005

Photodynamic therapy using 5-aminolevulinic acid–induced protoporphyrin IX synthesis as a photosensitizing reagent is an encouraging modality for cancer treatment. Understanding the mechanism of tumor phototoxicity is important to provide a basis for combinatory therapy regimens. A normal cell line (UROtsa, urothelial) and two tumor cell lines (RT4, urothelial; HT29, colonic) were treated with cell line–specific LD50 doses of light after exposure to 5-aminolevulinic acid (100 μg/mL), and harvested for RNA extraction 0, 10, and 30 minutes after irradiation. The RNA was hybridized to the metg001A Affymetrix GeneChip containing 2,800 genes, focusing on cancer-related and growth regulatory targets. Comparing the gene expression profiles between the different samples, 40 genes (e.g., SOD2, LUC7A, CASP8, and DUSP1) were identified as significantly altered in comparison with the control samples, and grouped according to their gene ontology. We selected caspase-8 (CASP8) and dual specificity phosphatase 1 (DUSP1) for further validation of the array findings, and compared their expression with the expression of the immediate early gene FOS by quantitative reverse transcription-PCR. RNA expression of CASP8 stayed unchanged whereas DUSP1 RNA was up-regulated in normal and tumor cells starting 30 minutes after irradiation. In contrast, FOS RNA was found continuously up-regulated over time in all three cell lines. Induction of DUSP1 protein expression was clearly shown after 1 hour using Western blot analysis. Interestingly, no changes of caspase-8 protein expression but activation of catalytic activity was detected only in UROtsa cells starting 1 hour after photodynamic therapy, whereas no changes were seen in both tumor cell lines. According to caspase-8, the active caspase 3 fragment was found only in the normal urothelial cell line (UROtsa) 1 hour after photodynamic therapy. Combined data analysis suggests that photodynamic therapy in vitro (LD50) leads to apoptosis in UROtsa and to necrosis in the tumor cell lines, respectively. RNA expression profiling of normal and tumor cell lines following photodynamic therapy with 5-aminolevulinic acid gave insight into the major molecular mechanisms induced by photodynamic therapy.
Selective induction of apoptosis in mutant p53 premalignant and malignant cancer cells by PRIMA-1 through the c-Jun-NH2-kinase pathway
Authors: Li Yin , Mao Yuehua , Brandt-Rauf Paul W. , Williams Ann C. , Fine Robert L.
Published: September 2005

PRIMA-1 (p53 reactivation and induction of massive apoptosis) is a chemical compound that was originally identified as a selective mutant p53-dependent growth suppressor by screening a library of low-molecular-weight compounds. However, its mechanism of action is unknown. In this study, we examined toxicity of PRIMA-1 to three premalignant human colorectal adenoma cell lines (RG/C2, BR/C1, and AA/C1) and four colorectal carcinoma cell lines (DLD-1, SW480, LOVO, and HCT116) and its mechanism of action. It selectively induced apoptosis only in the mutant p53 premalignant and malignant colon cell lines, but was not toxic to the wild-type p53 premalignant and malignant colon cell lines. Using stable transfectants of temperature-sensitive p53 mutant Ala143 in null p53 H1299 lung cancer cells, we found that PRIMA-1 induced significantly more apoptosis in cells with mutant p53 conformation (37°C) than the wild-type p53 conformation (32.5°C). Cell cycle analysis indicated that its inhibition of cell growth was correlated with induction of G2 arrest. Western blot analysis showed PRIMA-1 increased p21 and GADD45 expression selectively in the mutant p53 cells. However, Fas, Bcl-2 family proteins, and caspases were not involved in PRIMA-1–induced cell death. The c-Jun-NH2-kinase (JNK) inhibitor SP 600125, but not p38 mitogen-activated protein kinase inhibitor SB 203580 or extracellular signal-regulated kinase inhibitor PD 98059, blocked PRIMA-1–induced apoptosis. Transfection with a dominant-negative phosphorylation mutant JNK, but not a dominant-negative p38 or wild-type JNK, inhibited PRIMA-1–induced cell death, suggesting that the JNK pathway plays an important role in PRIMA-1–induced apoptosis. PRIMA-1 is a highly selective small molecule toxic to p53 mutant cells and may serve as a prototype for the development of new p53-targeting agents for therapy of premalignant and malignant cells.
A-432411, a novel indolinone compound that disrupts spindle pole formation and inhibits human cancer cell growth
Authors: Chen Zehan , Merta Philip J. , Lin Nan-Hong , Tahir Stephen K. , Kovar Peter , Sham Hing L. , Zhang Haiying
Published: September 2005

Microtubules are among the most successful targets for anticancer therapies and for the development of new anticancer drugs. A-432411 is a novel small molecule that destabilizes microtubules at high concentration and disrupts normal spindle formation at low concentration. A-432411 is an indolinone that is structurally different from other known synthetic microtubule inhibitors. This compound is efficacious against a variety of human cancer cell lines including drug-resistant HCT-15 that overexpresses Pgp170. Biochemical studies show that A-432411 competes with the colchicine-binding site on tubulin and inhibits microtubule polymerization. Fluorescence-activated cell sorting analysis indicates that A-432411 causes G2-M arrest and induces apoptosis. Cells treated with A-432411 have increased level of phospho-histone H3 at Ser10 and decreased level of phospho-cdc2 at Tyr15. Concurrently, securin and cyclin B1 expression levels remain the same, indicating the activation of the spindle checkpoint. Immunocytochemistry and fluorescence microscopy experiments reveal that 1 μmol/L A-432411 destabilizes microtubules in cells. At 0.1 μmol/L, the compound disrupts normal spindle pole formation possibly through stabilization of microtubule dynamic. Both structural and cellular properties of A-432411 make it an attractive candidate for further development.
Targeted molecular therapy of anaplastic thyroid carcinoma with AEE788
Authors: Kim Seungwon , Schiff Bradley A. , Yigitbasi Orhan G. , Doan Dao , Jasser Samar A. , Bekele B. Nebiyou , Mandal Mahitosh , Myers Jeffrey N.
Published: September 2005

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human malignancies with a mean survival of only 6 months. The poor prognosis of patients with ATC reflects the current lack of curative therapeutic options and the need for development of novel therapeutic strategies. In this study, we report the results of a preclinical study of AEE788, a dual inhibitor of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, against ATC. AEE788 was able to inhibit the proliferation and induce apoptosis of ATC cell lines in vitro. Administration of AEE788, alone and in combination with paclitaxel, to athymic nude mice bearing s.c. ATC xenografts inhibited the growth of ATC xenografts by 44% and 69%, respectively, compared with the control group. Furthermore, tumors from mice treated with AEE788, alone and in combination with paclitaxel, showed increase in apoptosis of tumor cells by ∼6- and 8-fold, respectively, compared with the control group. The microvessel density within the ATC xenografts was decreased by >80% in the mice treated with AEE788 alone and in combination with paclitaxel compared with the control group. Lastly, immunofluorescence microscopy showed the inhibition of EGFR autophosphorylation on the tumor cells as well as the inhibition of VEGFR-2 autophosphorylation on tumor endothelium. Considering the fact that curative options seldom exist for patients with ATC, concurrent inhibition of EGFR and VEGFR tyrosine kinases seems to be a valid and promising anticancer strategy for these patients.
Involvement of cathepsin D in chemotherapy-induced cytochrome c release, caspase activation, and cell death
Authors: Emert-Sedlak Lori , Shangary Sanjeev , Rabinovitz Asaf , Miranda Michelle B. , Delach Scott M. , Johnson Daniel E.
Published: September 2005

Treatment of cells with chemotherapy drugs activates the intrinsic mitochondrial pathway of apoptosis and the caspase protease cascade. Recently, the lysosomal protease cathepsin D has been implicated in apoptosis caused by oxidative stress, inhibition of protein kinase C, and stimulation of the TNFR1 and Fas death receptors. However, the role of cathepsin D in chemotherapy-induced cell death has remained largely unexplored. In this report, we show that treatment of U937 leukemia cells with the chemotherapy drug etoposide (VP-16) results in cathepsin D release into the cytosol within 4 hours after initiation of drug treatment. VP-16-induced cathepsin D release was not inhibited by z-VAD-FMK or pepstatin A, suggesting that it occurred independently of the activities of caspase proteases or cathepsin D. Down-regulation of cathepsin D expression in suspension U937 cells or adherent HeLa cells using cathepsin D small interfering RNA partially inhibited cell death resulting from treatment of cells with tumor necrosis factor-α, tumor necrosis factor–related apoptosis inducing ligand, or the chemotherapy drugs VP-16, cisplatin, and 5-fluorouracil. Moreover, cathepsin D down-regulation significantly delayed cytochrome c release and caspase-3 activation in response to chemotherapy treatment. Incubation of isolated mitochondria with cathepsin D–treated cytosolic extracts resulted in potent release of cytochrome c, indicating that a cytoplasmic substrate mediates the effects of cathepsin D on mitochondria. Together, these findings show that cathepsin D plays an important role in chemotherapy-induced cell death, and that cathepsin D lies upstream of cytochrome c release and caspase-3 activation in the chemotherapy-induced execution pathway.
Circulating plasma vascular endothelial growth factor in mice bearing human ovarian carcinoma xenograft correlates with tumor progression and response to therapy
Authors: Manenti Luigi , Riccardi Elena , Marchini Sergio , Naumova Elitza , Floriani Irene , Garofalo Angela , Dossi Romina , Marrazzo Eleonora , Ribatti Domenico , Scanziani Eugenio , Bani MariaRosa , Belotti Dorina , Broggini Massimo , Giavazzi Raffaella
Published: September 2005

Vascular endothelial growth factor (VEGF) performs as an angiogenic and permeability factor in ovarian cancer, and its overexpression has been associated with poor prognosis. However, models to study its role as a marker of tumor progression are lacking. We generated xenograft variants derived from the A2780 human ovarian carcinoma (1A9), stably transfected with VEGF121 in sense (1A9-VS-1) and antisense orientation (1A9-VAS-3). 1A9, 1A9-VS-1, and 1A9-VAS-3 disseminated in the peritoneal cavity of nude mice, but only 1A9-VS-1, the VEGF121-overexpressing tumor variant, produced ascites. Tumor biopsies from 1A9-VS-1 showed alterations in the vascular pattern and caused an angiogenic response in the chorioallantoic membrane assay. A significant level of soluble VEGF was detectable in the plasma of mice bearing 1A9-VS-1 even at an early stage of tumor growth. Plasma VEGF correlated positively with tumor burden in the peritoneal cavity and ascites accumulation. Cisplatin reduced the tumor burden and ascites in mice bearing 1A9-VS-1; the response was associated with a significant decrease of VEGF in plasma.

This 1A9-VS-1 xenograft model reproduces the behavior of human ovarian cancer by growing in the peritoneal cavity, being highly malignant, and producing ascites. Plasma VEGF as a marker of tumor progression offers a valuable means of detecting early tumor response and following up treatments in an animal model.
Small-interfering RNA–induced androgen receptor silencing leads to apoptotic cell death in prostate cancer
Authors: Liao Xinbo , Tang Siqing , Thrasher J. Brantley , Griebling Tomas L. , Li Benyi
Published: September 2005

Prostate cancer is the second leading cause of cancer death in the United States and, thus far, there has been no effective therapy for the treatment of hormone-refractory disease. Recently, the androgen receptor (AR) has been shown to play a critical role in the development and progression of the disease. In this report, we showed that knocking down the AR protein level by a small interfering RNA (siRNA) approach resulted in a significant apoptotic cell death as evidenced by an increased annexin V binding, reduced mitochondrial potential, caspase-3/6 activation, and DFF45 and poly(ADP-ribose) polymerase cleavage. The apoptotic response was specifically observed in those siRNA-transfected cells that harbor a native AR gene. No cell death was found in the AR-null prostate cancer cell PC-3 or its subline that has been reconstituted with an exogenous AR gene, as well as two breast cancer cell lines that are AR positive. Moreover, in parallel with the siRNA-induced AR silencing, the antiapoptotic protein Bcl-xL was significantly reduced, which might account for the apoptotic cell death because ectopic enforced expression of Bcl-xL protein partially inhibited apoptosis after AR silencing. Taken together, our data showed that knocking down the AR protein level in prostate cancer cells leads to apoptosis by disrupting the Bcl-xL–mediated survival signal downstream of AR-dependent survival pathway.
Irinotecan pharmacokinetic and pharmacogenomic alterations induced by methylselenocysteine in human head and neck xenograft tumors
Authors: Azrak Rami G. , Yu Jinsheng , Pendyala Lakshmi , Smith Patrick F. , Cao Shousong , Li Xia , Shannon William D. , Durrani Farukh A. , McLeod Howard L. , Rustum Youcef M.
Published: September 2005

The combination of methylselenocysteine and irinotecan (CPT-11) is synergistic against FaDu and A253 xenografts. Methylselenocysteine/CPT-11 increased tumor cure rate to 100% in FaDu and to 60% in A253. In this study, the effect of methylselenocysteine on pharmacokinetic and pharmacogenetic profiles of genes relevant to CPT-11 metabolic pathway was evaluated to identify possible mechanisms associated with the observed combinational synergy. Nude mice bearing tumors (FaDu and A253) were treated with methylselenocysteine, CPT-11, and a combination of methylselenocysteine/CPT-11. Samples were collected and analyzed for plasma and intratumor concentration of CPT-11 and 7-ethyl-10-hydroxyl-camptothecin (SN-38) by high-performance liquid chromatography. The intratumor relative expression of genes related to the CPT-11 metabolic pathway was measured by real-time PCR. After methylselenocysteine treatment, the intratumor area under the concentration-time curve of SN-38 increased to a significantly higher level in A253 than in FaDu and was associated with increased expression of CES1 in both tumors. Methylselenocysteine/CPT-11 treatment, compared with CPT-11 alone, resulted in a significant decrease in levels of ABCC1 and DRG1 in FaDu tumors and an increase in levels of CYP3A5 and TNFSF6 in A253 tumors. No statistically significant changes induced by methylselenocysteine/CPT-11 were observed in the levels of other investigated variables. In conclusion, the significant increase in the cure rate after methylselenocysteine/CPT-11 could be related to increased drug delivery into both tumors (CES1), reduced resistance to SN-38 (ABCC1 and DRG1) in FaDu, and induced Fas ligand apoptosis (TNFSF6) in A253. No correlation was observed between cure rate and other investigated variables (transporters, degradation enzymes, DNA repair, and cell survival/death genes) in either tumor.
Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer
Authors: Cao Carolyn , Shinohara Eric T. , Niermann Kenneth J. , Donnelly Edwin F. , Chen Xinping , Hallahan Dennis E. , Lu Bo
Published: September 2005

Murine double minute 2 (MDM2) inhibits p53-mediated functions, which are essential for therapies using DNA-damaging agents. The purpose of this study was to determine whether MDM2 inhibition enhances the radiosensitivity of a lung cancer model. The effects of MDM2 inhibition on tumor vasculature were also studied. Transient transfection of H460 lung cancer cells and human umbilical vascular endothelial cells (HUVEC) with antisense oligonucleotides (ASODN) against MDM2 resulted in a reduced level of MDM2 and increased levels of p21 and p53. Clonogenic assays showed that inhibition of MDM2 greatly decreased cell survival following irradiation. Quantification of apoptotic cells by 7-aminoactinomycin D staining and of senescent cells by X-gal staining showed that both processes were significantly increased in H460 cells treated with MDM2-specific ASODN and radiation. H460 xenografts that were treated with MDM2 ASODN plus radiotherapy also showed significant growth delay (P < 0.001) and increased apoptosis by terminal deoxynucleotidyl transferase–mediated nick end labeling staining. HUVECs transfected with MDM2-specific ASODN showed impaired viability and migration with decreased tube formation. Doppler studies showed that tumor blood flow was compromised when H460 xenografts were treated with MDM2-specific ASODN and radiation. A combination of radiotherapy and inhibition of MDM2 through the antisense approach results in improved tumor control in the H460 lung cancer model. This implies that a similar strategy should be investigated among patients with locally advanced lung cancer, receiving thoracic radiotherapy.
Tumor penetration of gefitinib (Iressa), an epidermal growth factor receptor tyrosine kinase inhibitor
Authors: McKillop David , Partridge Elizabeth A. , Kemp John V. , Spence Mike P. , Kendrew Jane , Barnett Sharon , Wood Phillippa G. , Giles Petrina B. , Patterson Andrew B. , Bichat Francis , Guilbaud Nicolas , Stephens Trevor C.
Published: September 2005

The relative distribution of gefitinib-related material in nude mice bearing s.c. human tumor xenografts and in an orthotopic rat lung tumor model was investigated following oral administration (50 mg/kg) of [14C]-gefitinib. Selected tissue samples were monitored for radioactivity by liquid scintillation counting, whereas plasma and tumor extracts were assayed for gefitinib and its major metabolites (M523595 and M537194) by high-performance liquid chromatography with tandem mass spectrometric detection. Tissue distribution was also determined by whole body autoradiography. Gefitinib was extensively distributed into the tissues of tumor-bearing mice and unchanged gefitinib was shown to account for most of the tumor radioactivity. Concentrations of gefitinib in mouse s.c. tumor xenografts were similar to skin concentrations and substantially greater (up to 12-fold based on area under the concentration-time curve) than plasma. Concentrations of gefitinib-related material in an orthotopic rat lung tumor were similar to those in healthy lung tissue and were much higher than corresponding blood levels. Following treatment of breast cancer patients with oral gefitinib (Iressa) 250 mg/d for ≥14 days, gefitinib concentrations (mean, 7.5 μg/g, 16.7 μmol/L) in breast tumor tissue were 42 times higher than plasma, confirming the preferential distribution of gefitinib from blood into tumor tissue in the clinical situation. These gefitinib tumor concentrations are considerably higher than those reportedly required in vitro to achieve complete inhibition of epidermal growth factor receptor autophosphorylation in both epidermal growth factor receptor mutant (0.2 μmol/L) and wild-type cells (2 μmol/L).
An evaluation of the ability of pifithrin-α and -β to inhibit p53 function in two wild-type p53 human tumor cell lines
Authors: Walton Mike I. , Wilson Stuart C. , Hardcastle Ian R. , Mirza Amin R. , Workman Paul
Published: September 2005

The small-molecule compound pifithrin-α (PFT-α) has been reported to inhibit p53 function and protect against a variety of genotoxic agents. We show here that PFT-α is unstable in tissue culture medium and is rapidly converted to its condensation product PFT-β. Both compounds showed limited solubility with PFT-α precipitating out of tissue culture medium at concentrations >30 μmol/L. PFT-α and -β exhibited cytotoxic effects in vitro towards two human wild-type p53–expressing tumor cell lines, A2780 ovarian and HCT116 colon (IC50 values for both cell lines were 21.3 ± 8.1 μmol/L for PFT-α and 90.3 ± 15.5 μmol/L for PFT-β, mean ± SD, n = 4). There was no evidence of protection by clonogenic assay with either compound in combination with ionizing radiation. Indeed, there was some evidence that PFT-α enhanced cytotoxicity, particularly at higher concentrations of PFT-α. Neither compound had any effect on p53, p21, or MDM-2 protein expression following ionizing radiation exposure and there was no evidence of any abrogation of p53-dependent, ionizing radiation–induced cell cycle arrest. Similarly, there was no evidence of cellular protection, or of effects on p53-dependent gene transcription, or on translation of MDM-2 or p21 following UV treatment of these human tumor cell lines. In addition, there was no effect on p53 or p21 gene transactivation or p38 phosphorylation after UV irradiation of NIH-3T3 mouse fibroblasts. In conclusion, neither PFT-α nor -β can be regarded as a ubiquitous inhibitor of p53 function, and caution should be exercised in the use of these agents as specific p53 inhibitors.
The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth
Authors: Jordan Mary Ann , Kamath Kathryn , Manna Tapas , Okouneva Tatiana , Miller Herbert P. , Davis Celia , Littlefield Bruce A. , Wilson Leslie
Published: September 2005

E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G2-M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/anaphase checkpoint.
Conditionally replicative adenovirus expressing degradation-resistant p53 for enhanced oncolysis of human cancer cells overexpressing murine double minute 2
Authors: van Beusechem Victor W. , van den Doel Petra B. , Gerritsen Winald R.
Published: September 2005

Conditionally replicative adenoviruses (CRAd) are under investigation as anticancer agents. Previously, we found that the CRAd AdΔ24-p53, expressing the p53 tumor suppressor protein from its genome, more effectively killed most human cancer cells than did its parent AdΔ24. However, a minority of cancer cell lines poorly responded to the oncolysis-enhancing effect of p53. Here we show that refractory cell lines expressed high levels of the major negative p53 regulator murine double minute 2 (MDM2). To obviate MDM2-mediated inactivation of CRAd-encoded p53, we constructed the new CRAd AdΔ24-p53(14/19) encoding a p53 variant incapable of binding to MDM2. AdΔ24-p53(14/19) was ∼10 times more effective than AdΔ24-p53 in killing cancer cell lines with high levels of human MDM2, but not cells with low MDM2. This finding supports the notion that exogenous expression of functional p53 augments the anticancer efficacy of CRAds. In addition, it confirms that high MDM2 expression is a molecular determinant of resistance against oncolysis enhancement by exogenous wild-type p53. Moreover, it shows that efficacy enhancement by restoration of functional p53 can also be accomplished in cancer cells expressing a p53 inhibitor. This further expands the utility of CRAds expressing functional p53 variants for effective virotherapy of cancer and thus their possible contribution to the advancement of individualized molecular medicine.
Potentiation of paclitaxel-induced apoptosis by the novel cyclin-dependent kinase inhibitor NU6140: a possible role for survivin down-regulation
Authors: Pennati Marzia , Campbell Allyson J. , Curto Maria , Binda Mara , Cheng YuZhu , Wang Lan-Zeng , Curtin Nicola , Golding Bernard T. , Griffin Roger J. , Hardcastle Ian R. , Henderson Andrew , Zaffaroni Nadia , Newell David R.
Published: September 2005

Cyclin-dependent kinases (CDK) play a crucial role in the control of the cell cycle. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, CDKs are promising targets for anticancer therapy. Here, we define the cellular effects of the novel CDK inhibitor NU6140, alone or in association with paclitaxel, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in HeLa cervical carcinoma cells and in comparison with purvalanol A. Both CDK inhibitors induced a concentration-dependent cell cycle arrest at the G2-M phase and an increase in the apoptotic rate, with a concomitant down-regulation of the antiapoptotic protein survivin, a member of the inhibitors of apoptosis protein family. Notably, the addition of NU6140 to paclitaxel-treated cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with the paclitaxel-purvalanol A combination (86 ± 11% and 37 ± 8%, respectively). Similarly, the extent of caspase-9 and caspase-3 activation in paclitaxel-NU6140–treated cells was ∼4-fold higher than after the paclitaxel-purvalanol A combination. Moreover, an almost complete abrogation of the expression of the active, Thr34-phosphorylated form of survivin was observed in cells exposed to the paclitaxel-NU6140 combination. A synergistic effect of the paclitaxel-NU6140 combination, as a consequence of survivin inhibition and increased activation of caspase-9 and caspase-3, was also observed in OAW42/e ovarian cancer line but not in the derived OAW42/Surv subline ectopically expressing survivin. Results from this study indicate that NU6140 significantly potentiates the apoptotic effect of paclitaxel, with inhibition of survivin expression/phosphorylation as the potential mechanism.
Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria
Authors: Wu Chin-Chung , Chan Mei-Ling , Chen Wen-Ying , Tsai Ching-Yi , Chang Fang-Rong , Wu Yang-Chang
Published: September 2005

Pristimerin, a naturally occurring triterpenoid, has been shown to cause cytotoxicity in several cancer cell lines. However, the mechanism for the cytotoxic effect of pristimerin was never explored. In the present study, human breast cancer MDA-MB-231 cells treated with pristimerin (1 and 3 μmol/L) showed rapid induction of apoptosis, as indicated by caspase activation, DNA fragmentation, and morphologic changes. Pretreatment of a pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) completely prevented pristimerin-induced apoptosis. Treatment of tumor cells with pristimerin resulted in a rapid release of cytochrome c from mitochondria, which preceded caspase activation and the decrease of mitochondrial membrane potential. In addition, neither benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone nor permeability transition pore inhibitor cyclosporin A markedly prevented pristimerin-induced mitochondrial cytochrome c release. Pristimerin did not significantly alter the protein level of Bcl-2 family members (Bcl-2, Bcl-XL, and Bax), nor did it induce Bax translocation. Moreover, Bcl-2 overexpression fails to prevent pristimerin-induced apoptosis. The generation of reactive oxygen species in MDA-MB-231 cells was also not affected by pristimerin. In a cell-free system, pristimerin induced cytochrome c release from isolated mitochondria. Taken together, these results suggested that pristimerin is a novel mitochondria-targeted compound and may be further evaluated as a chemotherapeutic agent for human cancer.