bluuebunny/crossref_metadata_2025 huggingface dataset:
Human
CYP1B1
Is Regulated by Estradiol via Estrogen Receptor
Authors: Tsuchiya Yuki , Nakajima Miki , Kyo Satoru , Kanaya Taro , Inoue Masaki , Yokoi Tsuyoshi
Published: September 2005

Human cytochrome P450 (CYP) 1B1 is a key enzyme in the metabolism of 17β-estradiol (E2). CYP1B1 is mainly expressed in endocrine-regulated tissues, such as mammary, uterus, and ovary. Because many CYP enzymes are likely to be induced by the substrates themselves, we examined whether the human CYP1B1 expression is regulated by E2 in the present study. Real-time reverse transcription-PCR analysis revealed that treatment with 10 nm E2 for 12 h induced CYP1B1 mRNA expression in estrogen receptor (ER)-positive MCF-7 cells. Luciferase reporter assays using MCF-7 cells showed a significant transactivation up to 7-fold by E2 with a reporter plasmid containing a region from −152 to +25 of the human CYP1B1 gene. A computer-assisted homology search indicated a putative estrogen response element (ERE) between −63 and −49 in the CYP1B1 promoter region. Specific binding of ERα to the putative ERE was demonstrated by chromatin immunoprecipitation assays and gel shift analyses. With reporter plasmids containing the wild or mutated putative ERE on the CYP1B1 gene and the wild or mutated ERα expression vectors, luciferase assays using Ishikawa cells demonstrated that the putative ERE and ERα are essential for the transactivation by E2. Because endometrial tissue is highly regulated by estrogens, the expression pattern of CYP1B1 protein in human endometrial specimens was examined by immunohistochemistry. The staining of CYP1B1 was stronger in glandular epithelial cells during a proliferative phase than those during a secretory phase, consistent with the pattern of estrogen secretion. These findings clearly indicated that the human CYP1B1 is regulated by estrogen via ERα. Because 4-hydroxylation of estrogen by CYP1B1 leads to decrease of the estrogenic activity but the produced metabolite is toxicologically active, our findings suggest a clinical significance in the estrogen-regulated CYP1B1 expression for the homeostasis of estrogens as well as estrogen-dependent carcinogenesis.
Two Distinct Pathways of Immuno-Modulation Improve Potency of p53 Immunization in Rejecting Established Tumors
Authors: Daftarian Pirouz , Song Guang-Yun , Ali Saima , Faynsod Moshe , Longmate Jeff , Diamond Don J. , Ellenhorn Joshua D. I.
Published: September 2005

The p53 gene product is overexpressed by almost 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated rejection of established p53-overexpressing tumors without stimulating autoimmunity by immunization with modified vaccinia Ankara-expressing murine p53 (MVAp53). Tumor rejection was enhanced through antibody-mediated CTL-associated antigen 4 (CTLA-4) blockade. We examined the role of synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODN) in enhancing MVAp53-mediated tumor rejection. CpG ODN with MVAp53 resulted in tumor rejection in BALB/c mice bearing poorly immunogenic 11A-1 murine mammary carcinomas or Meth A sarcomas and C57Bl/6 mice bearing MC-38 colon carcinomas. The effect was similar to that seen in tumor-bearing mice treated with MVAp53 along with CTLA-4 blockade. Monoclonal antibody depletion experiments demonstrated that the adjuvant effects of CpG ODN and CTLA-4 blockades were CD8 dependent. CpG ODN were partially natural killer cell dependent and ineffective in Toll-like Receptor 9−/− and interleukin 6−/− mice, whereas CTLA-4 blockade was partially CD4 dependent and functional in Toll-like Receptor 9−/− and interleukin 6−/− mice. In addition, when administered with MVAp53, both adjuvants enhanced p53-specific cytotoxicity and demonstrated an additive effect when combined. The combination of CpG ODN and CTLA-4 blockade worked synergistically to reject palpable 11A-1 and MC-38 tumors. These experiments demonstrate the potential for augmenting MVAp53-mediated antitumor immunity using CpG ODN and CTLA-4 blockade. This cell-free immunotherapy approach is a candidate for evaluation in cancer patients.
Frequent Silencing of
Low Density Lipoprotein Receptor-Related Protein 1B (LRP1B
) Expression by Genetic and Epigenetic Mechanisms in Esophageal Squamous Cell Carcinoma
Authors: Sonoda Itaru , Imoto Issei , Inoue Jun , Shibata Tatsuhiro , Shimada Yutaka , Chin Koei , Imamura Masayuki , Amagasa Teruo , Gray Joe W. , Hirohashi Setsuo , Inazawa Johji
Published: September 2005

Low-density lipoprotein receptor-related protein 1B (LRP1B) is frequently deleted in tumors of various types, but its status and expression in esophageal squamous cell carcinomas (ESCs) have never been reported. In the course of a program to screen ESC cell lines for copy-number aberrations using array-based comparative genomic hybridization, we identified a homozygous deletion of LRP1B. Genomic PCR experiments revealed homozygous deletions of LRP1B in additional ESC cell lines (total, 6 of 43; 14.0%) and in primary esophageal tumors (30 of 70; 42.9%). Moreover, expression of LRP1B mRNA was frequently silenced in ESC lines without homozygous deletions (14 of 37; 37.8%). Using bisulfite-PCR analysis and sequencing, we found that LRP1B-nonexpressing cells without homozygous deletions were highly methylated at a CpG island of LRP1B, a sequence possessing promoter activity. Treatment with 5-aza-2′-deoxycytidine restored expression of LRP1B in those ESC lines. Histone acetylation status correlated directly with expression of LRP1B and inversely with the methylation status of the CpG island. Methylation of LRP1B was also detected in primary esophageal tumors. Restoration of LRP1B expression in ESC cells reduced colony formation. These results suggest that loss of LRP1B function in esophageal carcinogenesis most often occurs either by homozygous deletion or by transcriptional silencing through hypermethylation of its CpG island.
Cyclooxygenase (COX)-2 Inhibitor Celecoxib Abrogates Activation of Cigarette Smoke-Induced Nuclear Factor (NF)-κB by Suppressing Activation of I-κB α Kinase in Human Non-Small Cell Lung Carcinoma
Authors: Shishodia Shishir , Aggarwal Bharat B.
Published: September 2005

Cigarette smoke (CS) has been linked to cardiovascular, pulmonary, and malignant diseases. CS-associated malignancies including cancers of the larynx, oral cavity, and pharynx, esophagus, pancreas, kidney, bladder, and lung; all are known to overexpress the nuclear factor-κB (NF-κB)-regulated gene products cyclin D1, cyclooxygenase (COX)-2, and matrix metalloprotease-9. Whether the COX-2 inhibitor, celecoxib, approved for the treatment of colon carcinogenesis and rheumatoid arthritis, affects CS-induced NF-κB activation is not known, although the role of NF-κB in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation is established. In our study, in which we examined DNA binding of NF-κB in human lung adenocarcinoma H1299 cells, we found that cigarette smoke condensate (CSC)-induced NF-κB activation was persistent up to 24 h, and celecoxib suppressed CSC-induced NF-κB activation. Celecoxib was effective even when administered 12 h after CSC treatment. This effect, however, was not cell type-specific. The activation of inhibitory subunit of NF-κB kinase (IκB), as examined by immunocomplex kinase assay, IκB phosphorylation, and IκB degradation was also inhibited. Celecoxib also abrogated CSC-induced p65 phosphorylation and nuclear translocation and NF-κB-dependent reporter gene expression. CSC-induced NF-κB reporter activity induced by NF-κB inducing kinase and IκB α kinase but not that activated by p65 was also blocked by celecoxib. CSC induced the expression of NF-κB-regulated proteins, COX-2, cyclin D1, and matrix metalloproteinase-9, and celecoxib abolished the induction of all three. The COX-2 promoter that is regulated by NF-κB was activated by CSC, and celecoxib suppressed its activation. Overall, our results suggest that chemopreventive effects of celecoxib may in part be mediated through suppression of NF-κB and NF-κB-regulated gene expression, which may contribute to its ability to suppress inflammation, proliferation, and angiogenesis.
Inhibition of Xenografted Human Melanoma Growth and Prevention of Metastasis Development by Dual Antiangiogenic/Antitumor Activities of Pigment Epithelium-Derived Factor
Authors: Garcia Marta , Fernandez-Garcia Nuria Isabel , Rivas Veronica , Carretero Marta , Escamez Maria J. , Gonzalez-Martin Alicia , Medrano Estela E. , Volpert Olga , Jorcano Jose L. , Jimenez Benilde , Larcher Fernando , Rio Marcela Del
Published: September 2005

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.
Overexpression of the Anti-Adhesin Podocalyxin Is an Independent Predictor of Breast Cancer Progression
Authors: Somasiri Aruna , Nielsen Julie S. , Makretsov Nikita , McCoy Marcia L. , Prentice Leah , Gilks C. Blake , Chia Stephen K. , Gelmon Karen A. , Kershaw David B. , Huntsman David G. , McNagny Kelly M. , Roskelley Calvin D.
Published: September 2005

Podocalyxin is a CD34-related cell surface molecule with anti-adhesive qualities. We probed a tissue microarray (n = 272) linked to long-term outcome data and found that podocalyxin was highly overexpressed in a distinct subset of invasive breast carcinomas (n = 15; 6%). Univariate disease-specific (P < 0.01) and multivariate regression (P < 0.0005) analyses indicated that this overexpression is an independent indicator of poor outcome. Forced podocalyxin expression perturbed cell junctions between MCF-7 breast carcinoma cells, and it caused cell shedding from confluent monolayers. Therefore, podocalyxin overexpression is a novel predictor of breast cancer progression that may contribute to the process by perturbing tumor cell adhesion.
Promoter Hypermethylation of
FANCF
Authors: Narayan Gopeshwar , Arias-Pulido Hugo , Nandula Subhadra V. , Basso Katia , Sugirtharaj Dorcas D. , Vargas Hernan , Mansukhani Mahesh , Villella Jeannine , Meyer Larissa , Schneider Achim , Gissmann Lutz , Dürst Matthias , Pothuri Bhavana , Murty Vundavalli V. V. S.
Published: September 2005

Patients with advanced stage invasive cervical cancer (CC) exhibit highly complex genomic alterations and respond poorly to conventional treatment protocols. In our efforts to understand the molecular genetic basis of CC, we examined the role of Fanconi Anemia (FA)-BRCA pathway. Here, we show that FANCF gene is disrupted by either promoter hypermethylation and/or deregulated gene expression in a majority of CC. Inhibition of DNA methylation and histone deacetylases induces FANCF gene re-expression in CC cell lines. FANCF-deregulated CC cell lines also exhibit a chromosomal hypersensitivity phenotype after exposure to an alkylating agent, a characteristic of FA patients. We also show the involvement of BRCA1 gene by promoter hypermethylation or down-regulated expression in a small subset of CC patients. Thus, we have found inactivation of genes in the FA-BRCA pathway by epigenetic alterations in a high proportion of CC patients, suggesting a major role for this pathway in the development of cervical cancer. Thus, these results have important implications in understanding the molecular basis of CC tumorigenesis and clinical management in designing targeted experimental therapeutic protocols.
Estrogen-Related Receptor α in Human Breast Carcinoma as a Potent Prognostic Factor
Authors: Suzuki Takashi , Miki Yasuhiro , Moriya Takuya , Shimada Norihiro , Ishida Takanori , Hirakawa Hisashi , Ohuchi Noriaki , Sasano Hironobu
Published: September 2005

Estrogen-related receptor α (ERRα) was identified as a gene related to estrogen receptor α (ERα) and belongs to a class of nuclear orphan receptors. ERRα binds to estrogen responsive element(s) (ERE) and is considered to be involved in modulation of estrogenic actions. However, biological significance of ERRα remains largely unknown. Therefore, we examined the expression of ERRα in human breast carcinoma tissues using immunohistochemistry (n = 102) and real-time reverse transcription-PCR (n = 30). ERRα immunoreactivity was detected in the nuclei of carcinoma cells in 55% of breast cancers examined, and relative immunoreactivity of ERRα was significantly (P = 0.0041) associated with the mRNA level. Significant associations were detected between ERα and ERE-containing estrogen-responsive genes, such as pS2 (P < 0.0001) and EBAG9/RCAS1 (P = 0.0214), in breast carcinoma tissues. However, no significant association was detected between ERα and pS2 (P = 0.1415) in the ERRα-positive cases (n = 56) or between ERα and EBAG9/RCAS1 (P = 0.8271) in the ERRα-negative group (n = 46). ERRα immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni- (P = 0.0097 and P = 0.0053, respectively) and multi- (P = 0.0215 and P = 0.0118, respectively) variate analyses. A similar tendency was also detected in the group of breast cancer patients who received tamoxifen therapy after surgery. Results from our study suggest that ERRα possibly modulates the expression of ERE-containing estrogen-responsive genes, and ERRα immunoreactivity is a potent prognostic factor in human breast carcinoma.
RASSF1A Interacts with Microtubule-Associated Proteins and Modulates Microtubule Dynamics
Authors: Dallol Ashraf , Agathanggelou Angelo , Fenton Sarah L. , Ahmed-Choudhury Jalal , Hesson Luke , Vos Michele D. , Clark Geoffrey J. , Downward Julian , Maher Eamonn R. , Latif Farida
Published: September 2005

The candidate tumor suppressor gene RASSF1A is inactivated in many types of adult and childhood cancers. However, the mechanisms by which RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel RASSF1A-interacting proteins in a human brain cDNA library. Seventy percent of interacting clones had homology to microtubule-associated proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A association with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed in mammalian cell lines. This suggested that RASSF1A may exert its tumor-suppressive functions through interaction with the microtubules. We demonstrate that RASSF1A associates with the microtubules, causing them to exist as hyperstabilized circular bundles. We found that two naturally occurring tumor-associated missense substitutions in the RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acetylation and were unable to protect the microtubules against the depolymerizing action of nocodazole. In addition, wild-type RASSF1A but not the C65R or the R257Q is able to block DNA synthesis. Our data identify a role for RASSF1A in the regulation of microtubules and cell cycle dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells.
Hypoxia Inhibits Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis by Blocking Bax Translocation
Authors: Kim Moonil , Park Sang-Youel , Pai Hyun-Sook , Kim Tae-Hyoung , Billiar Timothy R. , Seol Dai-Wu
Published: September 2005

The hypoxic environment in solid tumors results from oxygen consumption by rapid proliferation of tumor cells. Hypoxia has been shown to facilitate the survival of tumor cells and to be a cause of malignant transformation. Hypoxia also is well known to attenuate the therapeutic activity of various therapies in cancer management. These observations indicate that hypoxia plays a critical role in tumor biology. However, little is known about the effects of hypoxia on apoptosis, especially on apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a potent apoptosis inducer that has been shown to specifically limit tumor growth without damaging normal cells and tissues in vivo. To address the effects of hypoxia on TRAIL-induced apoptosis, HCT116 human colon carcinoma cells were exposed to hypoxic or normoxic conditions and treated with soluble TRAIL protein. Hypoxia dramatically inhibited TRAIL-induced apoptosis in HCT116 cells, which are highly susceptible to TRAIL in normoxia. Hypoxia increased antiapoptotic Bcl-2 family member proteins and inhibitors of apoptosis proteins. Interestingly, these hypoxia-increased antiapoptotic molecules were decreased by TRAIL treatment to the levels lower than those of the untreated conditions, suggesting that hypoxia inhibits TRAIL-induced apoptosis via other mechanisms rather than up-regulation of these antiapoptotic molecules. Additional characterization revealed that hypoxia significantly inhibits TRAIL-induced translocation of Bax from the cytosol to the mitochondria in HCT116 and A549 cells, with the concomitant inhibition of cytochrome c release from the mitochondria. Bax-deficient HCT116 cells were completely resistant to TRAIL regardless of oxygen content, demonstrating a pivotal role of Bax in TRAIL-induced apoptotic signaling. Thus, our data indicate that hypoxia inhibits TRAIL-induced apoptosis by blocking Bax translocation to the mitochondria, thereby converting cells to a Bax-deficient state.
Hath1, Down-Regulated in Colon Adenocarcinomas, Inhibits Proliferation and Tumorigenesis of Colon Cancer Cells
Authors: Leow Ching Ching , Romero Maria S. , Ross Sarajane , Polakis Paul , Gao Wei-Qiang
Published: September 2005

A striking feature of colon tumors is the significant reduction of goblet cells. Although targeted deletion of Math1 in mice leads to a loss of intestinal secretory cells, including goblet cells, the role of Hath1 in colon tumorigenesis remains unknown. Here we report that Hath1, the human ortholog of Math1, was dramatically down-regulated in colon tumor samples and colon cancer cell lines. Overexpression of Hath1 in HT29, an aggressive colon cancer cell line, resulted in a significant inhibition on cell proliferation, anchorage-independent growth in soft agar and, more importantly, growth of human colon cancer cell xenografts in athymic nude mice. Such inhibition was accompanied by altered expression of a goblet cell differentiation marker, MUC2, and cell cycle regulators cyclin D1 and p27kip1. Hath1 expression also was up-regulated on inhibition of the Wnt pathway, which has been well implicated in colon tumorigenesis. Hence, this study suggests that Hath1 may be a novel factor downstream of the Wnt pathway capable of suppressing anchorage-independent growth of colon cancer cell lines. More importantly, this study is the first to establish a link between down-regulation of Hath1 expression and colon tumorigenesis.
Deletion of Laminin-8 Results in Increased Tumor Neovascularization and Metastasis in Mice
Authors: Zhou Zhongjun , Doi Masayuki , Wang Jianming , Cao Renhai , Liu Baohua , Chan Kui Ming , Kortesmaa Jarkko , Sorokin Lydia , Cao Yihai , Tryggvason Karl
Published: September 2005

Laminin-8 (α4β1γ1) is one of the major laminin isoforms expressed in vascular endothelial basement membranes. Here we show that deletion of laminin-8 in mice affects angiogenesis under pathological conditions. Murine tumor models used in laminin α4-deficient mice results in hyperneovascularization and significant promotion of tumor growth and metastasis. The higher tumor growth rates in mutant mice correlate with decreased tumor cell apoptosis. Depletion of laminin α4 chain may alter the structure of vascular basement membranes, leading to increased angiogenesis. Our data suggest that the laminin-8 plays a critical role in the regulation of pathological angiogenesis.
Chemopreventive Effects of a Selective Nitric Oxide Synthase Inhibitor on Carcinogen-Induced Rat Esophageal Tumorigenesis
Authors: Chen Tong , Nines Ronald G. , Peschke Sarah M. , Kresty Laura A. , Stoner Gary D.
Published: September 2005

The inducible nitric oxide synthase (iNOS) generates a high concentration of nitric oxide (NO) in tissues. Increased NO production is associated with many disorders including esophageal cancer. Previous studies in our laboratory demonstrated an association between increased iNOS expression and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. On the basis of these observations, we initiated a bioassay to evaluate the ability of S,S′-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, to prevent the progression of esophageal tumors in rats preinitiated with NMBA. Rats were given s.c. injections of NMBA (0.25 mg/kg body weight) three times per week for 5 weeks. One week later, they were fed a synthetic diet containing either 50 or 100 ppm PBIT until the end of the bioassay (25 weeks). PBIT reduced the incidence of esophageal cancer from 96% in NMBA-treated rats to 83% and 77% (P < 0.05) in rats treated with 50 and 100 ppm PBIT, respectively. Tumor multiplicity was reduced from 3.64 ± 0.42 tumors per esophagus in NMBA-treated rats to 1.79 ± 0.25 (P < 0.001) and 1.50 ± 0.24 (P < 0.0001) in rats treated with 50 and 100 ppm PBIT, respectively. PBIT reduced the production of NO in NMBA-induced preneoplastic and papillomatous esophageal lesions when compared with comparable lesions in rats treated with NMBA only. iNOS mRNA expression was not modulated by PBIT. These observations suggest that iNOS plays a role in tumor development and that its selective inhibitor, PBIT, significantly inhibits esophageal tumor progression presumably through reducing the production of NO.
In vivo
Detection of Gastric Cancer in Rats by Electron Paramagnetic Resonance Imaging
Authors: Mikuni Tomiko , He Guanglong , Petryakov Sergey , Fallouh Mohanad M. , Deng Yuanmu , Ishihara Ryu , Kuppusamy Periannan , Tatsuta Masaharu , Zweier Jay L.
Published: September 2005

Electron paramagnetic resonance imaging (EPRI) enables noninvasive spatial mapping of free radical metabolism and has recently been shown to provide in vivo physiologic information regarding alterations in the redox state of tumors and neoplastic tissues. With the use of nitroxide spin probes, it has been shown that certain tumors possess a highly reduced state. To determine whether EPRI can be used for early detection and visualization of gastric carcinoma based on its altered redox metabolism, studies were performed in a rat gastric cancer model induced by 1-methyl-3-nitro-1-nitrosoguanidine. Using a specialized 750 MHz resonator and EPRI instrument, a technique was developed for imaging nitroxide radicals in the whole stomach. In vivo three-dimensional EPRI of the stomach of rats with continuous intravenous administration of nitroxide 3-carboxamido-2,2,5,5-tetramethylpyrrolidine-N-oxyl (3-carbamoyl-proxyl) [3-CP] was performed. Whereas electron paramagnetic resonance images from untreated controls provide a uniform visualization of the stomach mucosa and wall, in the treated rats with gastric cancer, holes were present in the image at the locations of tumors. With localized spectroscopy, it was confirmed that the tumor regions were devoid of signal, and this was largely due to the presence of a more reduced state with rapid reduction of nitroxide. Pharmacokinetic studies indicated that 3-CP in tumors was rapidly reduced to an undetectable level, whereas the 3-CP levels in normal stomach tissue persisted. Near-infrared reflectance measurements of indocyanine green dye uptake indicated that there were no significant differences in tumor versus normal mucosal perfusion. From these results, we concluded that gastric cancer tumors could be distinguished from normal tissue based primarily on the marked difference in their rate of radical metabolism. Because alterations in cellular redox state and radical metabolism are of critical importance in tumor biology and treatment, this methodology should provide an important new tool for the study and visualization of gastric carcinoma and may also be of use in other cancer models.
Diminished Hepatocellular Proliferation in Mice Humanized for the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α
Authors: Cheung Connie , Akiyama Taro E. , Ward Jerrold M. , Nicol Christopher J. , Feigenbaum Lionel , Vinson Charles , Gonzalez Frank J.
Published: September 2005

Lipid-lowering fibrate drugs function as agonists for the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Sustained activation of PPARα leads to the development of liver tumors in rats and mice. However, humans appear to be resistant to the induction of peroxisome proliferation and the development of liver cancer by fibrate drugs. The molecular basis of this species difference is not known. To examine the mechanism determining species differences in peroxisome proliferator response between mice and humans, a PPARα-humanized mouse line was generated in which the human PPARα was expressed in liver under control of the tetracycline responsive regulatory system. The PPARα-humanized and wild-type mice responded to treatment with the potent PPARα ligand Wy-14643 as revealed by induction of genes encoding peroxisomal and mitochondrial fatty acid metabolizing enzymes and resultant decrease of serum triglycerides. However, surprisingly, only the wild-type mice and not the PPARα-humanized mice exhibited hepatocellular proliferation as revealed by elevation of cell cycle control genes, increased incorporation of 5-bromo-2′-deoxyuridine into hepatocyte nuclei, and hepatomegaly. These studies establish that following ligand activation, the PPARα-mediated pathways controlling lipid metabolism are independent from those controlling the cell proliferation pathways. These findings also suggest that structural differences between human and mouse PPARα are responsible for the differential susceptibility to the development of hepatocarcinomas observed after treatment with fibrates. The PPARα-humanized mice should serve as models for use in drug development and human risk assessment and to determine the mechanism of hepatocarcinogenesis of peroxisome proliferators.
Telomeric Recombination in Mismatch Repair Deficient Human Colon Cancer Cells after Telomerase Inhibition
Authors: Bechter Oliver E. , Zou Ying , Walker William , Wright Woodring E. , Shay Jerry W.
Published: September 2005

The majority of human malignancies use telomerase to maintain telomere homeostasis. Antitelomerase therapy is therefore a promising approach for a cancer-specific therapy. The alternative lengthening of telomeres pathway (ALT) is a recombination-based, telomerase-independent mechanism of telomere length control. It is widely believed that ALT could be engaged when cancer cells escape from telomerase inhibition. However, no reports exist that would support this concept of therapy resistance. We inhibited telomerase in a human cancer cell line with a mismatch repair defect and observed a telomerase-independent, ALT-like telomere elongation. This is the first report of inducing a telomerase-independent telomere elongation in human cancer cells when telomerase is inhibited, thus describing a novel mechanism of resistance to antitelomerase therapy.
Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis
Authors: Schnaeker Eva-Maria , Ossig Rainer , Ludwig Thomas , Dreier Rita , Oberleithner Hans , Wilhelmi M. , Schneider Stefan W.
Published: September 2005

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave and degrade a wide spectrum of extracellular matrix components. By enhancing turnover of extracellular matrix, MMP activity is also known to play a key role in tumor cell invasion. Because extracellular protease activity requires efficient release of these proteases to the cellular surface, we investigated storage, transport, and exocytosis of MMP-2 and MMP-9 in human melanoma cells using immunofluorescence, electrical, and biochemical techniques. Immunolabeling of melanoma cells with antibodies specific for MMP-2 and MMP-9 led to the identification of two distinct populations of small cytoplasmatic vesicles containing MMP-2 or MMP-9, respectively. In combination with α-tubulin–specific antibodies, both vesicle populations were found to be aligned along the microtubular network. Moreover, the molecular motor protein kinesin is shown to be localized on most of these vesicles, providing evidence that the identified vesicles are actively propelled along microtubules toward the plasma membrane. The functional relevance of these findings is demonstrated using low dosage (5.9 nmol/L) of paclitaxel to affect the microtubular function of melanoma cells. Although cell proliferation is not altered, paclitaxel treatment impairs secretion of MMP-2/MMP-9 and significantly reduces invasive activity in our new cell invasion assay. In conclusion, we demonstrate in melanoma cells that microtubule-dependent traffic of MMP-containing vesicles and exocytosis are critical steps for invasive behavior and therefore are potential targets for specific antitumor drugs.
Endogenous Galectin-3 Determines the Routing of CD95 Apoptotic Signaling Pathways
Authors: Fukumori Tomoharu , Takenaka Yukinori , Oka Natsuo , Yoshii Tadashi , Hogan Victor , Inohara Hidenori , Kanayama Hiro-omi , Kim Hyeong-Reh Choi , Raz Avraham
Published: September 2005

Studies of CD95 (APO-1/Fas), a member of the death receptor family, have revealed that it is involved in two primary CD95 apoptotic signaling pathways, one regulated by the large amount of active caspase-8 (type I) formed at the death-inducing signaling complex and the other by the apoptogenic activity of mitochondria (type II). To date, it is still unclear which pathway will be activated in response to an apoptotic insult. Here, we demonstrate that the antiapoptotic molecule galectin-3, which contains the four amino acid-anti-death-motif (NWGR) conserved in the BH1 domain of the Bcl-2 member proteins, is expressed only in type I cells. Transfection of galectin-3 cDNA into galectin-3 null cells (type II) resulted converting them to type I apoptotic phenotype. In addition, we show that galectin-3 is complexed with CD95 in vivo identifying galectin-3 as a novel CD95-binding partner that determines which of the CD95 apoptotic signaling pathways the cell will select.
Akt/Protein Kinase B Signaling Inhibitor-2, a Selective Small Molecule Inhibitor of Akt Signaling with Antitumor Activity in Cancer Cells Overexpressing Akt
Authors: Yang Lin , Dan Han C. , Sun Mei , Liu Qiyuan , Sun Xia-meng , Feldman Richard I. , Hamilton Andrew D. , Polokoff Mark , Nicosia Santo V. , Herlyn Meenhard , Sebti Said M. , Cheng Jin Q.
Published: September 2005

Accumulated studies have shown that activation of the Akt pathway plays a pivotal role in malignant transformation and chemoresistance by inducing cell survival, growth, migration, and angiogenesis. Therefore, Akt is believed to be a critical target for cancer intervention. Here, we report the discovery of a small molecule Akt pathway inhibitor, Akt/protein kinase B signaling inhibitor-2 (API-2), by screening the National Cancer Institute Diversity Set. API-2 suppressed the kinase activity and phosphorylation level of Akt. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit the activation of phosphatidylinositol 3′-kinase, phosphoinositide-dependent kinase-1, protein kinase C, serum- and glucocorticoid-inducible kinase, protein kinase A, signal transducer and activators of transcription 3, extracellular signal-regulated kinase-1/2, or c-Jun NH2-terminal kinase. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/activated but not of those cancer cells in which it is not. These findings provide strong evidence for pharmacologically targeting Akt for anticancer drug discovery.
The Nuclear Receptor Coactivator AIB1 Mediates Insulin-like Growth Factor I-induced Phenotypic Changes in Human Breast Cancer Cells
Authors: Oh Annabell , List Heinz-Joachim , Reiter Ronald , Mani Aparna , Zhang Ying , Gehan Edmund , Wellstein Anton , Riegel Anna T.
Published: September 2005

The nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is overexpressed in human breast cancers and is required for estrogen signaling. However, the role of AIB1 in breast cancer etiology is not known. Here, we show that AIB1 is rate-limiting for insulin-like growth factor I (IGF-I)-dependent phenotypic changes and gene expression in human breast cancer cells. Reduction of endogenous AIB1 levels by small interfering RNA in MCF-7 breast cancer cells prevented IGF-I–stimulated anchorage-independent growth by reducing IGF-I–dependent anti-anoikis. cDNA array and immunoblot analysis of gene expression revealed that reduction in AIB1 levels led to a significant decrease in the expression of several genes controlling the cell cycle and apoptosis. These AIB1-dependent changes were also observed in the presence of estrogen antagonist and were corroborated in the estrogen receptor-negative cell line MDA MB-231. AIB1 reduction decreased the expression of the IGF-I receptor and IRS-1 in MCF-7 but not in MDA MB-231 cells. IGF-I–stimulated activation of AKT was reduced by AIB1 small interfering RNA treatment, whereas mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation by IGF-I was unaffected. We conclude that AIB1 is required for IGF-I–induced proliferation, signaling, cell survival, and gene expression in human breast cancer cells, independent of its role in estrogen receptor signaling.
Fenretinide Cytotoxicity for Ewing’s Sarcoma and Primitive Neuroectodermal Tumor Cell Lines Is Decreased by Hypoxia and Synergistically Enhanced by Ceramide Modulators
Authors: Batra Sandeep , Reynolds C. Patrick , Maurer Barry J.
Published: September 2005

Patients with disseminated Ewing’s family of tumors (ESFT) often experience drug-resistant relapse. We hypothesize that targeting minimal residual disease with the cytotoxic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR; fenretinide) may decrease relapse. We determined the following: (a) 4-HPR cytotoxicity against 12 ESFT cell lines in vitro; (b) whether 4-HPR increased ceramide species (saturated and desaturated ceramides); (c) whether physiological hypoxia (2% O2) affected cytotoxicity, mitochondrial membrane potential (ΔΨm) change, or ceramide species or reactive oxygen species levels; (d) whether cytotoxicity was enhanced by l-threo-dihydrosphingosine (safingol); (e) whether physiological hypoxia increased acid ceramidase (AC) expression; and (f) the effect of the AC inhibitor N-oleoyl-ethanolamine (NOE) on cytotoxicity and ceramide species. Ceramide species were quantified by thin-layer chromatography and scintillography. Cytotoxicity was measured by a fluorescence-based assay using digital imaging microscopy (DIMSCAN). Gene expression profiling was performed by oligonucleotide array analysis. We observed, in 12 cell lines tested in normoxia (20% O2), that the mean 4-HPR LC99 (the drug concentration lethal to 99% of cells) = 6.1 ± 5.4 μm (range, 1.7–21.8 μm); safingol (1–3 μm) synergistically increased 4-HPR cytotoxicity and reduced the mean 4-HPR LC99 to 3.2 ± 1.7 μm (range, 2.0–8.0 μm; combination index < 1). 4-HPR increased ceramide species in the three cell lines tested (up to 9-fold; P < 0.05). Hypoxia (2% O2) reduced ceramide species increase, ΔΨm loss, reactive oxygen species increase (P < 0.05), and 4-HPR cytotoxicity (P = 0.05; 4-HPR LC99, 19.7 ± 23.9 μm; range, 2.3–91.4). However, hypoxia affected 4-HPR + safingol cytotoxicity to a lesser extent (P = 0.04; 4-HPR LC99, 4.9 ± 2.3 μm; range, 2.0–8.2). Hypoxia increased AC RNA expression; the AC inhibitor NOE enhanced 4-HPR-induced ceramide species increase and cytotoxicity. The antioxidant N-acetyl-l-cysteine somewhat reduced 4-HPR cytotoxicity but did not affect ceramide species increase. We conclude the following: (a) 4-HPR was active against ESFT cell lines in vitro at concentrations achievable clinically, but activity was decreased in hypoxia; and (b) combining 4-HPR with ceramide modulators synergized 4-HPR cytotoxicity in normoxia and hypoxia.
CD44 Potentiates the Adherence of Metastatic Prostate and Breast Cancer Cells to Bone Marrow Endothelial Cells
Authors: Draffin Jayne E. , McFarlane Suzanne , Hill Ashleigh , Johnston Patrick G. , Waugh David J. J.
Published: September 2005

The aim of this current study was to examine the significance of CD44 expression in mediating cancer cell adhesion to human bone marrow endothelial cell(s) (hBMEC). Differential CD44 expression on two metastatic prostate cancer cell lines, PC3 (CD44 +ve) and DU145 (CD44 −ve) and four breast cancer cell lines was confirmed by immunoblotting and immunocytochemistry. In cell adhesion assays, PC3 but not DU145 cells demonstrated a rapid adhesion to hBMECs. Treatment of PC3 cells with a neutralizing antibody against CD44 standard (CD44s) and CD44 splice variants decreased PC3 cell adhesion to hBMECs. Similarly, depletion of CD44 expression using RNA interference decreased the ability of PC3 cells and two CD44 +ve breast cancer cell lines (MDA-MB-231 and MDA-MB-157) to bind FITC-conjugated hyaluronan (FITC-HA) and to adhere to hBMECs. In contrast, transfection of DU145 cells or the T47D and MCF-7 breast cancer cell lines to express CD44s increased cell surface binding of FITC-HA and cell adherence to hBMECs. Treatment of PC3 and MDA-MD-231 cells but not hBMECs with hyaluronidase attenuated cell adhesion, suggesting that cell surface expression of CD44 on prostate and breast cancer cells may promote the retention of a HA coat that facilitates their initial arrest on bone marrow endothelium.
Involvement of Ras Activation in Human Breast Cancer Cell Signaling, Invasion, and Anoikis
Authors: Eckert Lynn B. , Repasky Gretchen A. , Ülkü Aylin S. , McFall Aidan , Zhou Hong , Sartor Carolyn I. , Der Channing J.
Published: September 2005

Although mutated forms of ras are not associated with the majority of breast cancers (<5%), there is considerable experimental evidence that hyperactive Ras can promote breast cancer growth and development. Therefore, we determined whether Ras and Ras-responsive signaling pathways were activated persistently in nine widely studied human breast cancer cell lines. Although only two of the lines harbor mutationally activated ras, we found that five of nine breast cancer cell lines showed elevated active Ras-GTP levels that may be due, in part, to HER2 activation. Unexpectedly, activation of two key Ras effector pathways, the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and phosphatidylinositol 3′-kinase/AKT signaling pathways, was not always associated with Ras activation. Ras activation also did not correlate with invasion or the expression of proteins associated with tumor cell invasion (estrogen receptor α and cyclooxygenase 2). We then examined the role of Ras signaling in mediating resistance to matrix deprivation-induced apoptosis (anoikis). Surprisingly, we found that ERK and phosphatidylinositol 3′-kinase/AKT activation did not have significant roles in conferring anoikis resistance. Taken together, these observations show that Ras signaling exhibits significant cell context variations and that other effector pathways may be important for Ras-mediated oncogenesis, as well as for anoikis resistance, in breast cancer. Additionally, because ERK and AKT activation are not strictly associated with Ras activation, pharmacological inhibitors of these two signaling pathways may not be the best approach for inhibition of aberrant Ras function in breast cancer treatment.
1,1-Bis(3′-indolyl)-1-(
p
-substitutedphenyl)methanes Induce Peroxisome Proliferator-Activated Receptor γ-Mediated Growth Inhibition, Transactivation, and Differentiation Markers in Colon Cancer Cells
Authors: Chintharlapalli Sudhakar , Smith Roger , Samudio Ismael , Zhang Wei , Safe Stephen
Published: September 2005

1,1-Bis(3′indolyl)-1–(p-substitutedphenyl)methanes containing p-trifluoromethyl (DIM-C-pPhCF3), p-t-butyl (DIM-C-pPhtBu), and p-phenyl (DIM-C-pPhC6H5) groups induce peroxisome proliferator-activated receptor γ (PPARγ)-mediated transactivation in HT-29, HCT-15, RKO, and SW480 colon cancer cell lines. Rosiglitazone also induces transactivation in these cell lines and inhibited growth of HT-29 cells, which express wild-type PPARγ but not HCT-15 cells, which express mutant (K422Q) PPARγ. In contrast, DIM-C-pPhCF3, DIM-C-pPhtBu, and DIM-C-pPhC6H5 inhibited growth of both HT-29 and HCT-15 cells with IC50 values ranging from 1 to 10 μmol/L. Rosiglitazone and diindolylmethane (DIM) analogues did not affect expression of cyclin D1, p21, or p27 protein levels or apoptosis in HCT-15 or HT-29 cells but induced keratin 18 in both cell lines. However, rosiglitazone induced caveolins 1 and 2 in HT-29 but not HCT-15 cells, whereas these differentiation markers were induced by DIM-C-pPhCF3 and DIM-C-pPhC6H5 in both cell lines. Because overexpression of caveolin 1 is known to suppress colon cancer cell and tumor growth, the growth inhibitory effects of rosiglitazone and the DIM compounds are associated with PPARγ-dependent induction of caveolins.
Breast Cancer Risk and the DNA Double-Strand Break End-Joining Capacity of Nonhomologous End-Joining Genes Are Affected by
BRCA1
Authors: Bau Da-Tian , Fu Yi-Ping , Chen Shou-Tung , Cheng Ting-Chih , Yu Jyh-Cherng , Wu Pei-Ei , Shen Chen-Yang
Published: September 2005

A tumorigenic role of the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double-strand breaks (DSBs) has been suggested by the finding of a significant association between increased breast cancer risk and a cooperative effect of single nucleotide polymorphisms (SNPs) in NHEJ genes. However, the lack of an association between hereditary breast cancer and defective NHEJ genes prevents conclusions from being drawn about a link between NHEJ and breast cancer development. Recently, BRCA1-deficient mouse embryonic fibroblasts were found to have significantly reduced NHEJ activity, suggesting an accessory role of BRCA1 in NHEJ. The present study was performed to confirm this observation in human breast cancer cell lines and to examine whether the interaction between BRCA1 and NHEJ was of tumorigenic significance. Support for this hypothesis came from the findings that (a) a case-control study (469 breast cancer patients and 740 healthy controls) showed that the breast cancer risk associated with high-risk genotypes of NHEJ genes was significantly modified by the BRCA1 genotype. A significant increase in the cancer risk associated either with harboring one additional putative high-risk NHEJ genotype or with the joint effect of having reproductive risk factors (reflected by an interval of ≥12 years between menarche and first full-term pregnancy) and a higher number of high-risk genotypes of the NHEJ genes was only seen in women with at least one variant BRCA1 allele (i.e., the Glu/Gly or Gly/Gly forms of BRCA1 Glu1038Gly); and (b) a phenotype-based study measuring in vitro and in vivo NHEJ capacity showed that the precise end-joining capacity was different in breast cancer cell lines with different BRCA1 statuses being higher in BRCA1-expressing MCF-7 cells than in HCC1937 cells (defective BRCA1 expression). Furthermore, this end-joining capacity was decreased in MCF-7 cells in which BRCA1 expression was blocked using small interfering RNA and increased in HCC1937 transfected with full-length BRCA1. Because BRCA1 is a well-documented breast cancer susceptibility gene, this association between NHEJ and BRCA1 not only suggests a role of BRCA1 in NHEJ but also provides essential support for the tumorigenic contribution of NHEJ in breast cancer development.
Apo2 Ligand/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Cooperates with Chemotherapy to Inhibit Orthotopic Lung Tumor Growth and Improve Survival
Authors: Jin Hongkui , Yang Renhui , Fong Sharon , Totpal Klara , Lawrence David , Zheng Zhong , Ross Jed , Koeppen Hartmut , Schwall Ralph , Ashkenazi Avi
Published: September 2005

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a tumor necrosis factor superfamily member that induces apoptosis through the death receptors DR4 and/or DR5 in various cancer cell types but not in most normal cells. Several lung cancer cell lines express DR4 and DR5 and undergo apoptosis in vitro in response to Apo2L/TRAIL. We investigated the efficacy of recombinant soluble human Apo2L/TRAIL and its interaction with chemotherapy in xenograft models based on human NCI-H460 non-small cell lung carcinoma cells. In vitro, Taxol enhanced caspase activation and apoptosis induction by Apo2L/TRAIL. In vivo, Apo2L/TRAIL or Taxol plus carboplatin chemotherapy partially delayed progression of established subcutaneous tumor xenografts, whereas combined treatment caused tumor regression and a substantially longer growth delay. Apo2L/TRAIL, chemotherapy, or the combination of both inhibited growth of preformed orthotopic lung parenchymal tumors versus control by 60%, 57%, or 97%, respectively (all P < 0.01; n = 8–10). Furthermore, combination treatment improved day-90 survival relative to control (7 of 15 versus 1 of 15; P = 0.0003 by Mantel-Cox) as well as to Apo2L/TRAIL (3 of 14; P = 0.031) or chemotherapy (3 of 15; P = 0.035). These studies provide evidence for in vivo activity of Apo2L/TRAIL against lung tumor xenografts and underscore the potential of this ligand for advancing current lung cancer treatment strategies.
Anticachectic Effects of Formoterol
Authors: Busquets Sílvia , Figueras Maria T. , Fuster Gemma , Almendro Vanessa , Moore-Carrasco Rodrigo , Ametller Elisabet , Argilés Josep M. , López-Soriano Francisco J.
Published: September 2005

In cancer cachexia both cardiac and skeletal muscle suffer an important protein mobilization as a result of increased proteolysis. Administration of the β2-agonist formoterol to both rats and mice bearing highly cachectic tumors resulted in an important reversal of the muscle-wasting process. The anti-wasting effects of the drug were based on both an activation of the rate of protein synthesis and an inhibition of the rate of muscle proteolysis. Northern blot analysis revealed that formoterol treatment resulted in a decrease in the mRNA content of ubiquitin and proteasome subunits in gastrocnemius muscles; this, together with the decreased proteasome activity observed, suggest that the main anti-proteolytic action of the drug may be based on an inhibition of the ATP-ubiquitin-dependent proteolytic system. Interestingly, the β2-agonist was also able to diminish the increased rate of muscle apoptosis (measured as DNA laddering as well as caspase-3 activity) present in tumor-bearing animals. The present results indicate that formoterol exerted a selective, powerful protective action on heart and skeletal muscle by antagonizing the enhanced protein degradation that characterizes cancer cachexia, and it could be revealed as a potential therapeutic tool in pathologic states wherein muscle protein hypercatabolism is a critical feature such as cancer cachexia or other wasting diseases.