bluuebunny/crossref_metadata_2025 huggingface dataset:
The selective retinoid X receptor agonist bexarotene (LGD1069, Targretin) prevents and overcomes multidrug resistance in advanced breast carcinoma
Authors: Yen Wan-Ching , Lamph William W.
Published: September 2005

Acquired drug resistance represents a major challenge in the therapeutic management of breast cancer patients. We reported previously that the retinoid X receptor–selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating animal models of tamoxifen-resistant breast cancer. The goal of this study was to evaluate the effect of bexarotene on development of acquired drug resistance and its role in overcoming acquired drug resistance in advanced breast cancer. Paclitaxel, doxorubicin, and cisplatin were chosen as model compounds to determine the effect of bexarotene on the development of acquired drug resistance. Human breast cancer cells MDA-MB-231 were repeatedly treated in culture with a given therapeutic agent with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. Compared with parental cells, cells treated with a single therapeutic agent became resistant to the therapeutic agent, whereas cells treated with the bexarotene combination remained chemosensitive. Cells with acquired drug resistance, when treated with the combination, showed increased sensitivity to the cytotoxic agent. Furthermore, cells treated with the combination regimen had reduced invasiveness and angiogenic potential than their resistant counterparts. These in vitro findings were further confirmed in an in vivo MDA-MB-231 xenograft model. Our results suggest a role for bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced breast carcinoma.
Overexpression of hRFI (human ring finger homologous to inhibitor of apoptosis protein type) inhibits death receptor–mediated apoptosis in colorectal cancer cells
Authors: Konishi Tsuyoshi , Sasaki Shin , Watanabe Toshiaki , Kitayama Joji , Nagawa Hirokazu
Published: September 2005

The acquisition of antiapoptotic properties is one of the essential mechanistic steps in colorectal carcinogenesis and is closely correlated with a loss of chemosensitivity and radiosensitivity. Human ring finger homologous to inhibitor of apoptosis protein type (hRFI) is a newly discovered gene encoding a ring finger domain highly homologous to that of X chromosome–linked inhibitor of apoptosis protein. Immunohistochemistry has revealed that the expression of hRFI increased in transition from normal colorectal mucosas to adenomas and from adenomas to carcinomas, suggesting an essential role in the early stage of colorectal carcinogenesis. However, the function role of hRFI in colorectal carcinoma has not been elucidated. To determine whether hRFI possesses an antiapoptotic function in colorectal cancer cells, HCT116 colorectal cancer cells stably overexpressing hRFI were established. The hRFI transfectant exhibited significant resistance to apoptosis induced by tumor necrosis factor-α or tumor necrosis factor–related apoptosis-inducing ligand compared with control. This antiapoptotic response was associated with decreased activity of caspase-3, -8, and -9. We also established an antisense down-regulation of hRFI, which effectively reversed the antiapoptotic activity of the hRFI transfectant. This confirmed that the antiapoptotic property of the hRFI transfectant was not due to the clonal effect but in fact dependent on hRFI function. In conclusion, hRFI possesses an antiapoptotic function in HCT116 colorectal cancer cells. Considering the progressive increase of hRFI expression in the advance of the colorectal adenoma-carcinoma sequence, hRFI is one of the important players in colorectal carcinogenesis through its effect on apoptosis regulation.
A highly epothilone B–resistant A549 cell line with mutations in tubulin that confer drug dependence
Authors: Yang Chia-Ping Huang , Verdier-Pinard Pascal , Wang Fang , Lippaine-Horvath Eva , He Lifeng , Li Dansu , Höfle Gerhard , Ojima Iwao , Orr George A. , Horwitz Susan Band
Published: September 2005

A 95-fold epothilone B (EpoB)–resistant, but not dependent, A549 human lung carcinoma cell line, A549.EpoB40 (EpoB40), has a Gln to Glu mutation at residue 292 that is situated near the M-loop of βI-tubulin. Further selection of this cell line with higher concentrations of EpoB produced A549.EpoB480 (EpoB480), which is ∼900-fold resistant to EpoB. This cell line, like EpoB40, exhibits cross-resistance to Taxol and extreme sensitivity to vinblastine, but in contrast to EpoB40 it is unusually dependent on EpoB, requiring a minimum of 125 nmol/L EpoB to maintain normal growth. Sequence analysis of the β-tubulin and Kα1-tubulin genes in EpoB480 showed that, in addition to the β292 mutation, β60 was mutated from Val to Phe and α195 was mutated from Leu to Met. Mass spectrometry indicated that both the Val60Phe and Leu195Met mutations in βI- and Kα1-tubulin, respectively, were expressed at the protein level. Molecular modeling indicated that β60 is located at the end of the H1-S2 loop that has been implicated as a principal partner of the M-loop for contacts between protofilaments. A mutation at β60 could inhibit the lateral contacts between protofilaments, thereby destabilizing microtubules. α195 is located at the external surface of the microtubule that has been proposed as the domain that interacts with a variety of endogenous proteins, such as stathmin and microtubule-associated protein 4. A mutation at α195 could modulate the interactions between tubulin and regulatory proteins. We propose that the βVal60Phe mutation plays a critical role in the drug-dependent phenotype of EpoB480 cells.
Identification of novel targeting peptides for human ovarian cancer cells using “one-bead one-compound” combinatorial libraries
Authors: Aina Olulanu H. , Marik Jan , Liu Ruiwu , Lau Derick H. , Lam Kit S.
Published: September 2005

Using “one-bead one-compound” combinatorial chemistry technology, we generated random peptide libraries containing millions of 90 μm TentaGel beads, each with its own unique amino acid sequence. A cyclic random 8-mer library was screened with CAOV-3 (a human ovarian adenocarcinoma cell line) and beads with a unique ligand that bind to the cell surface receptors were coated by one or more layers of cells. These positive beads were isolated, stripped, and microsequenced. Several peptide motifs were identified from these screenings, some of which were novel and unique, e.g., cDGX4GX6X7c. Structure-activity relationship studies of this peptide revealed that the l-aspartate residue at position 2, the two glycines at positions 3 and 5, and the two d-cysteines at the amino and COOH terminus are critical for activity. In addition, a hydrophobic residue was preferred at position X4, whereas amino acids at positions X6 and X7 were more variable. Binding of this peptide to a number of different cancer cell lines and normal cells was also determined and we observed that peptides with this motif bound preferentially to three other human ovarian cancer cell lines (ES-2, SKOV-3, and OVCAR-3) as well as a human glioblastoma cancer cell line (A172). Structural analysis of the peptides using high-resolution nuclear magnetic resonance spectroscopy revealed strong conformational similarity among all peptides with cX1GX4GX6X7c motif. Blocking study with a panel of anti-integrin antibodies strongly suggests α3 integrin present on these ovarian adenocarcinoma cells is the target receptor for this peptide.
p37 induces tumor invasiveness
Authors: Ketcham Catherine M. , Anai Satoshi , Reutzel Robbie , Sheng Shijie , Schuster Sheldon M. , Brenes Ryan B. , Agbandje-McKenna Mavis , McKenna Robert , Rosser Charles J. , Boehlein Susan K.
Published: September 2005

Previous studies have shown a statistically significant correlation between human carcinomas and monoclonal antibody detection of a Mycoplasma hyorhinis–encoded protein known as p37. A potential mechanism of p37 is that it might promote invasion and metastasis. Recombinant p37 enhanced the invasiveness of two prostate carcinoma and two melanoma cell lines in a dose-dependent manner in vitro, but did not have a significant effect on tumor cell growth. Furthermore, the increased binding to cell surfaces and the enhanced invasive potential of cancer cells from exposure to p37 could be completely reversed by preincubation of the cancer cells with an anti-p37 monoclonal antibody. Sequence comparisons, followed by three-dimensional molecular modeling, revealed a region of similarity between p37 and influenza hemagglutinin A, a sialic acid–binding protein that plays a critical role in viral entry. Binding of p37 to prostate carcinoma cells was found to be at least partially sialic acid dependent because neuraminidase treatment decreased this binding. Taken together, these observations suggest that M. hyorhinis can infect humans and may facilitate tumor invasiveness via p37. These results further suggest that p37 may be a molecular target for cancer therapy.
Mitochondria are the primary target in isothiocyanate-induced apoptosis in human bladder cancer cells
Authors: Tang Li , Zhang Yuesheng
Published: September 2005

Many isothiocysanates (ITC) are promising cancer-preventive agents, and induction of apoptosis is one of their underlying mechanisms of action. We recently found that caspase-9 was preferentially activated over other initiator caspases in human bladder cancer UM-UC-3 cells. We report here that caspase-9 activation is the major step leading to ITC-induced apoptosis in this cell line. More importantly, our results show that caspase-9 activation by the ITCs may result primarily from mitochondrial damage. Four common naturally occurring ITCs were studied, including allyl ITC, benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane. BITC and PEITC showed more potent mitochondria-damaging ability than the other two ITCs, correlating well with their stronger apoptosis-inducing potentials. Furthermore, BITC and PEITC damaged both the outer and inner mitochondrial membranes. Use of isolated mitochondria allowed us to establish that ITCs, and more importantly their major intracellular derivatives (glutathione conjugates) at concentrations that are readily achievable in cells, damage mitochondria, leading to the collapse of mitochondrial trans-membrane potential and release of cytochrome c. The mitochondria-damaging potencies of the ITCs correlate well with their lipophilicities. Bcl-2 family members are known to influence the stability of mitochondrial membrane. Our results show that the ITCs caused phosphorylation of Bcl-2, induced mitochondrial translocation of Bak, and disrupted the association of Bcl-xl with both Bak and Bax in mitochondrial membrane, indicating that ITC-induced mitochondrial damage results at least in part from modulation of select Bcl-2 family members.
Multidrug-resistant neuroblastoma cells are responsive to arsenic trioxide at both normoxia and hypoxia
Authors: Karlsson Jenny , Edsjö Anders , Påhlman Sven , Pettersson Helen M.
Published: September 2005

Despite intensive treatment, the outcome of high-risk neuroblastoma patients is poor with acquired multidrug resistance as an important cause. Previously, our group has shown that arsenic trioxide (As2O3) kills multidrug-resistant neuroblastoma cells in vitro and in vivo at clinically tolerable doses. Regions of tissue hypoxia often arise in aggressive solid tumors, and hypoxic tumors exhibit augmented invasiveness and metastatic ability in several malignancies. Furthermore, hypoxia may impair the treatment efficiency; therefore, we have studied the cytotoxic effect of As2O3 on neuroblastoma cells grown under normoxic as well as hypoxic (1% oxygen) conditions. At both normoxia and hypoxia, 2 and 4 μmol/L As2O3 induced evident cell death in the drug-sensitive SH-SY5Y and IMR-32 cells as well as in the multidrug-resistant SK-N-BE(2)c (with a mutated p53) and SK-N-FI cells after 72 hours of exposure. In contrast, the conventional chemotherapeutic drug etoposide showed lowered efficiency in hypoxic IMR-32 cells. In accordance with our previously published results, although not to the same extent as in their normoxic counterparts, Bax is proteolytically cleaved also in neuroblastoma cells exposed to As2O3 at hypoxia. This suggests that similar molecular mechanisms are involved in As2O3-induced neuroblastoma cell death during hypoxia compared with normoxia. Together, our results support As2O3 as a potential candidate drug as a complement to conventional treatments for high-risk neuroblastoma patients and perhaps also for patients with other multidrug-resistant solid tumors.
Inhibition of insulin-like growth factor-I receptor (IGF-IR) signaling and tumor cell growth by a fully human neutralizing anti–IGF-IR antibody
Authors: Wang Yan , Hailey Judith , Williams Denise , Wang Yaolin , Lipari Philip , Malkowski Michael , Wang Xiaoying , Xie Lei , Li Guanghua , Saha Deba , Ling Wai Lam W. , Cannon-Carlson Susan , Greenberg Robert , Ramos Robert A. , Shields Robert , Presta Leonard , Brams Peter , Bishop W. Robert , Pachter Jonathan A.
Published: September 2005

Insulin-like growth factor-I receptor (IGF-IR) plays an important role in tumor cell growth and survival. On ligand stimulation, IGF-IR, a receptor tyrosine kinase, phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT pathways. Here, we describe the characterization of a fully human anti–IGF-IR monoclonal antibody 19D12 that inhibits IGF binding and autophosphorylation of both IGF-IR/IGF-IR homodimers and IGF-IR/insulin receptor heterodimers. 19D12 does not recognize insulin receptor homodimers. In addition to inhibiting IGF-IR autophosphorylation, 19D12 also inhibits IRS-1 phosphorylation and activation of the major downstream signaling molecules AKT and extracellular signal-regulated kinase 1/2. Furthermore, the antibody down-regulates the total IGF-IR protein level and can exhibit antibody-dependent cellular cytotoxicity activity against a non–small cell adenocarcinoma cell line in vitro in the presence of isolated human natural killer cells. 19D12 binds tightly to the receptor, with an affinity of 3.8 pmol/L as measured by KinExA. In cell culture, 19D12 inhibits proliferation and soft agar growth of various tumor cell lines. In vivo, 19D12 inhibits the tumor growth of a very aggressive human ovarian tumor xenograft model A2780. These data support the development of this anti–IGF-IR monoclonal antibody as a promising anticancer agent.
Low-dose cisplatin protects human neuroblastoma SH-SY5Y cells from paclitaxel-induced apoptosis
Authors: Villa Daniela , Miloso Mariarosaria , Nicolini Gabriella , Rigolio Roberta , Villa Antonello , Cavaletti Guido , Tredici Giovanni
Published: September 2005

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin α, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.
Hedamycin, a DNA alkylator, induces γH2AX and chromosome aberrations: Involvement of phosphatidylinositol 3-kinase–related kinases and DNA replication fork movement
Authors: Tu Lan Chun , Matsui Sei ichi , Beerman Terry A.
Published: September 2005

Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)–regulated phosphorylation of H2AX (γH2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX (γH2AX) foci, and chromosome aberrations. Hedamycin treatment of HCT116 carcinoma cells resulted in a rapid induction of DNA strand breaks accompanied by increasing H2AX phosphorylation and focalization. Unlike many other treatments that also stall replication, such as UV, camptothecin, and adozelesin, γH2AX formation was not suppressed in ATR-compromised cells but actually increased. Similarly, hedamycin induction of γH2AX is not dependent on ataxia telangiectasia mutated or DNA-protein kinase, and pretreatment of cells with the phosphatidylinositol 3-kinase–related kinase inhibitor caffeine did not substantially reduce induction of H2AX phosphorylation by hedamycin. Furthermore, the DNA replication inhibitor aphidicolin only modestly depressed hedamycin-induced γH2AX formation, indicating that hedamycin-induced DNA double-strand breaks are not dependent on fork progression. In contrast, camptothecin- and adozelesin-induced γH2AX was strongly suppressed by aphidicolin. Moreover, after 24 hours following a short-term hedamycin treatment, cells displayed high levels of breaks in interphase nuclear DNA and misjoined chromosomes in metaphase cells. Finally, focalization of a tightly bound form of Ku80 was observed in interphase cells, consistent with the subsequent appearance of chromosomal aberrations via abnormal nonhomologous end joining. Overall, this study has revealed a disparate type of DNA damage response to stalled replication induced by a bulky DNA adduct inducer, hedamycin, that seems not to be highly dependent on ATR or DNA replication.
Modulation of intratumoral hypoxia by the epidermal growth factor receptor inhibitor gefitinib detected using small animal PET imaging
Authors: Solomon Benjamin , Binns David , Roselt Peter , Weibe Leonard I. , McArthur Grant A. , Cullinane Carleen , Hicks Rodney J.
Published: September 2005

Blockade of signaling through the epidermal growth factor receptor (EGFR) tyrosine kinase by inhibitors such as gefitinib (Iressa) can inhibit tumor angiogenesis and enhance responses to ionizing radiation. In this study, the ability of gefitinib to modulate intratumoral oxygenation was evaluated in human EGFR-expressing A431 squamous cell carcinoma xenografts using in vivo small animal positron emission tomography (PET) imaging with the hypoxia marker [18F]fluoroazomycin arabinoside (FAZA) and by the immunohistochemical detection of hypoxia-induced adducts of the 2-nitroimidazole, pimonidazole. Serial noninvasive PET imaging of A431 xenografts showed a significant reduction in FAZA uptake following treatment with 75 mg/kg/d of gefitinib [tumor to background ratio, 6.1 ± 1.0 (pretreatment) versus 2.3 ± 0.6 (posttreatment); P = 0.0004]. Similarly, ex vivo quantitation of FAZA uptake showed significantly reduced FAZA uptake in established A431 xenografts treated with gefitinib compared with vehicle control (tumor to blood ratio for controls versus gefitinib, 8.0 ± 3.0 versus 2.7 ± 0.8; P = 0.007; or tumor to muscle ratio controls versus gefitinib, 8.6 ± 2.8 versus 2.6 ± 1.0; P = 0.002). The effect of gefitinib treatment seemed to be independent of tumor size. In addition, gefitinib treatment reduced pimonidazole-binding in A431 xenografts measured after 5 and 8 days of gefitinib treatment compared with baseline and with tumors treated with vehicle alone. A strong correlation was observed between pimonidazole binding and FAZA uptake. Together, these findings show that gefitinib reduces intratumoral hypoxia.
Synergistic enhancement of TRAIL- and tumor necrosis factor α–induced cell death by a phenoxazine derivative
Authors: Hara Keiichi , Okamoto Mayumi , Aki Toshihiko , Yagita Hideo , Tanaka Hirotoshi , Mizukami Yoichi , Nakamura Hiroshi , Tomoda Akio , Hamasaki Naotaka , Kang Dongchon
Published: September 2005

2-Amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) has been developed as a novel phenoxazine derivative having an anticancer activity on a variety of cancer cell lines as well as transplanted tumors in mice with minimal toxicity to normal cells. We examined the effects of Phx-1 on Jurkat cells, a human T cell line. Phx-1 inhibited proliferation of the cells in a dose-dependent manner but hardly induced cell death, suggesting that Phx-1 acts primarily as an antiproliferative reagent but not as a cytocidal drug. Phx-1 enhanced tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death about 100-fold. Tumor necrosis factor α, which alone does not induce cell death of Jurkat cells, caused apoptosis in combination with Phx-1. These enhancements of cell death were not due to up-regulation of the death receptors. Phx-1 decreased serum-induced phosphorylation of Akt, a kinase involved in cell proliferation and survival, and inhibited complex III of mitochondrial respiratory chain. Considering that both TRAIL and Phx-1 have only marginal cytotoxicity to most normal cells, Phx-1 may provide an ideal combination for cancer therapy with TRAIL.
Integrin-linked kinase is a potential therapeutic target for anaplastic thyroid cancer
Authors: Younes Maher N. , Kim Seungwon , Yigitbasi Orhan G. , Mandal Mahitosh , Jasser Samar A. , Dakak Yazici Yasemin , Schiff Bradley A. , El-Naggar Adel , Bekele Benjamin N. , Mills Gordon B. , Myers Jeffrey N.
Published: September 2005

We investigated integrin-linked kinase (ILK), a focal adhesion serine-threonine protein kinase, as a new molecular target for treating anaplastic thyroid cancer. ILK mediates cell growth and survival signals and is overexpressed in a number of cancers. Therefore, we hypothesized that inhibition of ILK leads to growth arrest and apoptosis of thyroid cancer cells. According to Western blotting, the level of ILK protein was highly expressed in one papillary (NPA187) and four of five (Hth74, DRO, ARO, KAT4, and K4) anaplastic thyroid cancer cell lines. Immunohistochemical analysis of a human tissue microarray revealed that ILK was highly expressed in anaplastic thyroid cancer but not in normal human thyroid tissue. Treating thyroid cancer cell lines with a new ILK inhibitor, QLT0267, inhibited epidermal growth factor–induced phosphorylation of AKT, inhibited cell growth, and induced apoptosis in the NPA187, DRO, and K4 cell lines. QLT0267 also inhibited the kinase activity of immunoprecipitated ILK in four of five cell lines. Tumor volumes in mice treated with QLT0267 were significantly reduced compared with those in untreated mice. In immunohistochemical studies, QLT0267 suppressed phosphorylated p-AKT and angiogenesis (i.e., reduced mean vascular density) and induced apoptosis in both tumor cells and tumor-associated endothelial cells of the thyroid DRO xenografts. In summary, we found that ILK expression and activity were elevated in human anaplastic thyroid cancer and ILK inhibition led to growth arrest and apoptosis in vitro and in vivo. Our results provide preliminary evidence that ILK is a potential therapeutic target for treating anaplastic thyroid cancer.
Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein
Authors: Voelkel-Johnson Christina , Hannun Yusuf A. , El-Zawahry Ahmed
Published: September 2005

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that selectively induces apoptosis in malignant cells. However, not all cancer cells are susceptible to TRAIL and mechanisms of resistance and new strategies to enhance sensitivity are an area of intense investigation. Glucose withdrawal or paclitaxel increase intracellular ceramide, down-regulate cellular FLICE inhibitory protein (cFLIP), and sensitize cells to TRAIL. Therefore, we investigated whether TRAIL resistance is due to ceramide levels and/or defects in ceramide generation following ligand binding. Colon cancer cells isolated from the primary tumor (SW480) and a subsequent metastasis (SW620) of the same patient have different sensitivities to TRAIL. Mass spectrometry was used to compare ceramide content in untreated and TRAIL-treated cells. Overall levels of ceramide were comparable in the cell lines but TRAIL-sensitive SW480 cells contained a higher percentage of C16-, and C18-ceramide and lower C24-ceramides than TRAIL-resistant SW620 cells. Upon TRAIL treatment, ceramide (primarily C16-ceramide) increased in SW480 but not SW620 cells. The increase in ceramide occurred with slow kinetics, paralleling caspase-3/7 activation. Combination of C6-ceramide with TRAIL resulted in apoptosis of SW620 cells. However, exogenous C6-ceramide did not affect levels of cFLIP nor did pretreatment sensitize cells to TRAIL. Exposure to TRAIL prior to ceramide was required to induce apoptosis, suggesting that ceramide plays a role in enhancing or amplifying TRAIL-mediated signaling. Our results suggest that ceramide plays a role in promoting TRAIL-mediated apoptosis and that TRAIL-resistant cancers may benefit from combination therapy with ceramide or agents that enhance ceramide accumulation.
Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930
Authors: Petti Filippo , Thelemann April , Kahler Jen , McCormack Siobhan , Castaldo Linda , Hunt Tony , Nuwaysir Lydia , Zeiske Lynn , Haack Herbert , Sullivan Laura , Garton Andrew , Haley John D.
Published: September 2005

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3′ kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non–receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.
Increased expression of the large subunit of ribonucleotide reductase is involved in resistance to gemcitabine in human mammary adenocarcinoma cells
Authors: Jordheim Lars Petter , Guittet Olivier , Lepoivre Michel , Galmarini Carlos M. , Dumontet Charles
Published: September 2005

Resistance to cytotoxic nucleoside analogues is a major problem in cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. However, this resistance seems to be multifactorial and more studies are needed to gain better insight into this domain. For this purpose, we developed a gemcitabine-resistant cell line (MCF7 1K) from the human mammary adenocarcinoma MCF7 strain by prolonged exposure to gemcitabine in vitro. MCF7 1K cells are highly resistant to gemcitabine (533-fold) and cross-resistance is observed with araC (47-fold), triapine (14-fold), and hydroxyurea (6.7-fold). Quantitative real-time reverse transcription-PCR and Western blot analysis showed an increase in the gene and protein expression of the large subunit of ribonucleotide reductase, R1. Ribonucleotide reductase activity was also significantly increased in the gemcitabine-resistant cells. Study of genomic DNA showed 12-fold increase in R1 gene dosage in MCF7 1K cells. In contrast, the gene and protein expression of the small subunit of ribonucleotide reductase, R2, were not modified in this cell line. These results show that gemcitabine resistance can be associated with genetic modifications of target genes in malignant cells, and suggest that the large subunit of human ribonucleotide reductase is involved in the cellular response to gemcitabine.
Competitive disruption of the tumor-promoting function of membrane type 1 matrix metalloproteinase/matrix metalloproteinase-14 in vivo
Authors: Nonaka Takahiro , Nishibashi Kunika , Itoh Yoshifumi , Yana Ikuo , Seiki Motoharu
Published: September 2005

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a potent modulator of the pericellular environment and promotes tumor cell invasion and proliferation in many types of tumor. The activation of proMMP-2 and processing of collagen I by MT1-MMP have been thought to be important for its tumor-promoting function. These activities can be inhibited by mutant forms of MT1-MMP lacking the catalytic domain. However, the effect of such dominant-negative mutants has never been evaluated in vivo. Various mutants lacking the catalytic domain (dCAT) were prepared and confirmed to inhibit MT1-MMP activity in human fibrosarcoma HT1080 cells, and tumor cells expressing these mutants were implanted s.c. into nude mice to monitor tumor formation. Only the membrane-anchored form of a dCAT construct through the transmembrane domain [dCAT(1)] showed potent antitumor activity not only in HT1080 cells but also in gastric carcinoma MKN28 and MKN45 cells expressing MT1-MMP. A soluble form of dCAT lacking the transmembrane domain did not show such activity. The expression of dCAT(1) in MKN28 or MKN45 further prevented the metastatic spread of tumor cells into the peritoneal cavity; however, dCAT(1) showed no effect against TMK-1, another gastric carcinoma cell line expressing no MT1-MMP. It is of note that the tumorigenicity of TMK-1 cells enhanced by MT1-MMP overexpression was, in turn, canceled by the additional expression of dCAT(1). Thus, MT1-MMP expressed in tumor cells seems to play a pivotal role in tumor growth in mice. The results also suggest new possibilities to abrogate the tumor-promoting function of MT1-MMP other than the conventional protease inhibitor–based approach.
Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair–deficient malignant glioma xenograft
Authors: Cheng C. Lynn , Johnson Stewart P. , Keir Stephen T. , Quinn Jennifer A. , Ali-Osman Francis , Szabo Csaba , Li Hongshan , Salzman Andrew L. , Dolan M. Eileen , Modrich Paul , Bigner Darell D. , Friedman Henry S.
Published: September 2005

Temozolomide is a DNA-methylating agent used in the treatment of malignant gliomas. In this study, we have examined if inhibition of poly(ADP-ribose) polymerase (PARP) could increase the cytotoxicity of temozolomide, particularly in cells deficient in DNA mismatch repair. Athymic mice, transplanted with mismatch repair–proficient [D-245 MG] or deficient [D-245 MG (PR)] xenografts, were treated with a combination of temozolomide and the PARP inhibitor, INO-1001. For the tumors deficient in mismatch repair, the most effective dose of INO-1001 was found to be 150 mg/kg, given i.p. thrice at 4-hour intervals with the first injection in combination with 262.5 mg/kg temozolomide (0.75 LD10). This dose of temozolomide by itself induced no partial regressions and a 4-day growth delay. In two separate experiments, the combination therapy increased the growth delay by 21.6 and 9.7 days with partial regressions observed in four of eight and three of nine mice, respectively. The addition of INO-1001 had a more modest, yet statistically significant, increase in tumor growth delay in the mismatch repair–proficient xenografts. In these experiments, mice were treated with a lower amount of temozolomide (88 mg/kg), which resulted in growth delays of 43.1 and 39.2 days. When the temozolomide treatment was in combination with 200 mg/kg INO-1001, there was an increase in growth delay to 48.9 and 45.7 days, respectively. These results suggest that inhibition of PARP may increase the efficacy of temozolomide in the treatment of malignant gliomas, particularly in tumors deficient in DNA mismatch repair.
Cyclooxygenase-independent down-regulation of multidrug resistance–associated protein-1 expression by celecoxib in human lung cancer cells
Authors: Kang He-Kyung , Lee Eunmyong , Pyo Hongryull , Lim Soo-Jeong
Published: September 2005

The recent finding of a link between cyclooxygenase-2 (COX-2) and p-glycoprotein expression suggests that COX-2 is involved in the development of the multidrug resistance (MDR) phenotype. MDR-associated protein 1 (MRP1) is another major MDR-related protein that is frequently overexpressed in cancer patients, including those with lung cancer. Based on our observation that among four human epithelial lung cell lines both MRP1 and COX-2 protein were highly expressed only in A549 cells, we have investigated whether COX-2 regulates the expression of MRP1. The COX-2 inhibitor celecoxib down-regulated the expression of MRP1 protein in A549 cells, which was accompanied by increased accumulation and enhanced cytotoxicity of doxorubicin, an MRP1 substrate. However, enforced expression of COX-2 in human H460 lung carcinoma cell lines, which express minimal level of COX-2, did not cause enhancement in MRP1 expression. Celecoxib down-regulation of MRP1 was observed independent of COX-2 expression. Moreover, in COX-2-overexpressing cell lines, celecoxib down-regulation of MRP1 was observed only at a concentration far exceeding that required for inhibiting COX activity, and exogenous addition of prostaglandin E2 did not restore MRP1 expression. These results suggest that celecoxib down-regulates MRP1 expression in human lung cancer cells in a COX-independent manner. The use of celecoxib for adjuvant therapy in lung cancer patients may contribute to their decreased resistance to chemotherapeutic drugs transported by MRP1.
Quantitative and Spatial Measurements of Telomerase Reverse Transcriptase Expression within Normal and Malignant Human Breast Tissues
Authors: Hines William C. , Fajardo Alexandra M. , Joste Nancy E. , Bisoffi Marco , Griffith Jeffrey K.
Published: September 2005

The enzyme telomerase catalyzes the de novo synthesis of telomere repeats, thereby maintaining telomere length, which is necessary for unlimited cellular proliferation. Telomerase reverse transcriptase (TERT), the catalytic domain of telomerase, is the rate-limiting factor for telomerase activity and is expressed in virtually all tumors. Thus, TERT has been proposed as a marker with diagnostic and prognostic potential in breast cancer as well as a basis for breast cancer therapeutics. In these contexts, it is important to define the sites and extent of TERT expression in normal and cancerous human breast tissues. In this study, levels of TERT mRNA were measured within a set of 36 breast carcinomas and 5 normal breast samples by quantitative real-time reverse transcription-PCR, and we subsequently identified and characterized the cells expressing TERT mRNA within these tissues using in situ hybridization. The results show that (a) detectable TERT mRNA expression is specific to the epithelial cells; (b) TERT is expressed in both normal and malignant breast tissues; (c) the pattern and level of TERT expression are heterogeneous, with ∼75% of tumors expressing bulk TERT mRNA levels equal to or less than those within normal breast tissue; and (d) tumors expressing above-normal levels of TERT mRNA are more likely to be histopathologic grade 3 (P = 0.002), contain high fraction of cells in S phase (P = 0.004), and have increased levels of MYC mRNA (P = 0.034).
The Current State of Cancer Gene Therapy and Its Application in Esophageal Carcinoma
Authors: Buskens Christianne J. , Marsman Willem A. , Bosma Piter J. , van Lanschot J. Jan B.
Published: September 2005

Advances in molecular genetics have accelerated the understanding of the genetic basis of many diseases. This is particularly true for esophageal adenocarcinoma with its well-defined premalignant lesions. At the same time, remarkable progress in recombinant DNA technology has enabled the development of molecular treatments for inherited disorders, infectious diseases and cancer. In recent years, especially the development of gene therapy systems as new treatment or prevention strategies for various malignant diseases has been explored. The present article deals with the general principles of gene therapy and then focuses on how these principles can be applied to esophageal cancer. Subsequently, the scarcely available experimental data from the literature are briefly reviewed. Finally, we summarize our experimental work over the last few years. Our main goal has been to develop an efficient and selective gene delivery system in order to maximize the proportion of successfully transduced tumor cells while sparing normal cells. Conclusion: The field of cancer gene therapy has evolved tremendously, with promising developments especially in the field of targeting and novel therapeutic genes. However, substantial research is still needed before gene therapy can play a significant role in the management of esophageal cancer. The focus of this research should be on further developing adequate experimental models as well as on improving gene-delivering vectors. Although it is unlikely that cancer gene therapy will replace the conventional methods of treatment, selective and efficient vectors may be used for patients with Barrett’s esophagus or as adjuvant therapy for patients with esophageal cancer in the future.
Liver Support Therapy: An Overview of the AMC-Bioartificial Liver Research
Authors: van de Kerkhove M.P. , Poyck P.P.C. , Deurholt T. , Hoekstra R. , Chamuleau R.A.F.M. , van Gulik T.M.
Published: September 2005

Acute liver failure (ALF) is a disease with a mortality of 60–90% depending on the cause. Only high-urgency liver transplantation is able to increase survival compared to standard intensive care therapy. Liver transplantation is hampered by the increasing shortage of organ donors, resulting in a high incidence of patients with ALF dying on the transplantation waiting list. Amongst a variety of liver assist therapies, bioartificial liver (BAL) therapy is marked as the most promising solution to bridge ALF patients to liver transplantation or to liver regeneration, since several BAL systems showed significant improvement of survival time in experimental animals with irreversible ALF. One of these systems has been developed at the Academic Medical Center in Amsterdam, The Netherlands – the AMC-BAL. This overview describes the development of the AMC-BAL based on porcine hepatocytes which was started 10 years ago. Positive results of in vitrofunctionality and in vivo safety and efficacy led to a successful phase I study in 12 ALF patients in Italy. However, xenotransplantation legislation in many European countries prohibits the use of porcine hepatocytes in clinically applied BAL systems. The future of the BAL, therefore, resides in the development of a human-derived hepatocyte cell line as biocomponent of BAL systems.
Minimizing Blood Loss in Liver Transplantation: Progress through Research and Evolution of Techniques
Authors: de Boer Marieke T. , Molenaar I. Quintus , Hendriks Herman G.D. , Slooff Maarten J.H. , Porte Robert J.
Published: September 2005

Blood loss during liver transplantation has long been recognized as an important cause of morbidity and, especially in the early days, also mortality. It is well known that blood transfusions are associated with an increased risk of postoperative complications, such as infections, pulmonary complications, protracted recovery, and a higher rate of reoperations. Many studies have been performed during the past decades to elucidate the mechanisms of increased blood loss in liver transplantation. In the late 1980s, primary hyperfibrinolysis was identified as an important mechanism of bleeding during liver transplantation. This has provided the scientific basis for the use of antifibrinolytic drugs in liver transplant recipients. Several randomized, controlled studies have shown the efficacy of these compounds in reducing blood loss and transfusion requirements during liver transplantation. In addition, increasing experience and improvements in surgical technique, anesthesiological care and better graft preservation methods have contributed to a steady decrease in blood transfusion requirements in most liver transplant programs. Several centers are now reporting liver transplantation without any need for blood transfusion in up to 30% of their patients. Despite these improvements, most patients undergoing liver transplantation still require blood transfusions that have a negative impact on outcome, emphasizing the need for further attempts to control blood loss by surgeons and anesthesiologists. This paper provides an overview of the clinical and research developments, which have contributed to a reduction in blood loss and transfusion requirements, resulting in an important reduction in morbidity and mortality after liver transplantation during the last two decades.
The Cyclin-Dependent Kinase Inhibitor p21 Limits Murine Mesangial Proliferative Glomerulonephritis
Authors: Monkawa Toshiaki , Pippin Jeffrey , Yo Yoshikage , Kopp Jeffrey B. , Alpers Charles E. , Shankland Stuart J.
Published: September 2005

Background: Mesangial cell (MC) proliferation underlies increased matrix accumulation in glomerulonephritis (GN), and the resolution of MC proliferation occurs largely through apoptosis. Proliferation and apoptosis are controlled by specific cell cycle proteins, where cyclin-dependent kinase (CDK) inhibitors such as p21 bind target cyclin-CDK complexes. However, the role of p21 in acute mesangial proliferative GN is not known. This study was conducted to test the hypothesis that p21 regulates MC proliferation and apoptosis in anti-MC serum-induced GN. Methods: Age and sex matched wild-type (p21+/+) and p21-deficient (p21–/–) mice were injected with sheep anti-MC serum. Renal function (BUN, urinary albumin excretion), histology, DNA synthesis (BrdU. Ki-67) and apoptosis (TUNEL) were quantified at day 6 and day 12 (n = 6–8/time point). Results: In p21+/+ mice, anti-MC-serum induced mild MC proliferative GN, and glomerular p21 expression was increased. Renal function was worse in nephric p21–/– mice. PAS and silver staining revealed that p21–/– mice had typical features of MC proliferative GN with focal segmental tuft necrosis, focal mesangiolysis and focal mesangial hypercellularity. Occasional features of podocyte injury (swelling, vacuolization) were noted. Double immunostaining confirmed increased mesangial cell DNA synthesis in nephritic p21–/– mice at day 6. In contrast, there was no difference in glomerular apoptosis in nephritic p21+/+ and p21–/– mice at each time point. Glomerular lesions were accompanied by severe glomerular and tubulointerstitial fibrosis in p21–/– mice. Conclusions: This data shows that the CDK-inhibitor p21 regulates the MC proliferative response to immune-mediated injury. In contrast, p21 does not alter the apoptotic response, resulting in a delayed resolution in nephritic p21–/– mice.
Male Castration Increases Neuronal Nitric Oxide Synthase Activity in the Rat Mesenteric Artery through Protein Kinase C Activation
Authors: Blanco-Rivero Javier , Balfagón Gloria , Ferrer Mercedes
Published: September 2005

The objective of the present study was to assess the effect of endogenous male sex hormones on the activity of protein kinase C (PKC), as well as the regulatory effect of this kinase on the neuronal nitric oxide (NO) release induced by electrical field stimulation (EFS; 200 mA; 0.3 ms; 1–16 Hz). For this purpose, superior mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. PKC activity was greater in arteries from orchidectomized than control rats. Basal and EFS-induced NO release was similar in arteries from both groups despite the lower nNOS expression in arteries from orchidectomized rats. Phorbol 12,13-dibutyrate (PDBu), a PKC activator, EFS-induced NO release was higher in arteries from control compared to orchidectomized rats. Calphostin C, a non-selective PKC inhibitor, or Gö6976, a PKC inhibitor partially selective for conventional isoforms,abolished the EFS-induced NO release in arteries from control animals, while it was decreased in arteries from orchidectomized animals. The PKCζ pseudosubstrate inhibitor decreased EFS-induced NO release equally in both groups. The NO synthase (NOS) inhibitor Nω-nitro-L-arginine-methyl ester (L-NAME) enhanced the EFS-elicited contractions in arteries from both groups. Calphostin C increased the contractions elicited by EFS in arteries from control and orchidectomized rats. This increase was further enhanced by calphostin C plus L-NAME only in orchidectomized rats. PDBu reduced EFS-induced contraction in arteries from controls but did not affect it in orchidectomized rats. The further addition of L-NAME increased the responses in both types of arteries. These results show that PKC activity is enhanced in mesenteric arteries from orchidectomized rats, which may be the responsible for the greater nNOS activity in these arteries. Conventional and atypical PKCζ isoforms positively regulate nNOS activity in arteries from both control and orchidectomized rats, but the contribution of conventional PKC isoforms to enhanced nNOS activity seems to be greater in arteries from orchidectomized rats.
Malignant Pleural/Pericardial Effusion with Tamponade and Life-Threatening Reversible Myocardial Depression in a Case of an Initial Presentation of Lung Adenocarcinoma
Authors: Ma Tony S. , Hayes Teresa G. , Levine Glenn N. , Carabello Blase A.
Published: September 2005

We present a case of a middle-aged woman in cardiac tamponade. Following pericardiocentesis that removed 1,500 ml of hemorrhagic fluid, the patient exhibited cardiogenic shock; LVEF, at its nadir, on inotrope, was less than 20%. Ventricular function slowly improved, with inotropic support, to the normal range by the 25th day of hospitalization. Cardiac failure in malignancy has often been attributed to multi-system failure; this case showed a hereto unrecognized clinical phenomenon – ‘malignancy-associated myopericarditis’. While the direct link of cause and effect cannot be made with certainty, the case should be instructive to other clinicians who encounter similar life-threatening presentations of cardiac decompensation in malignancy.