bluuebunny/crossref_metadata_2025 huggingface dataset:
Cancer Therapy with a Replicating Oncolytic Adenovirus Targeting the Hypoxic Microenvironment of Tumors
Authors: Post Dawn E. , Devi Narra Sarojini , Li Zhenchao , Brat Daniel J. , Kaur Balveen , Nicholson Ainsley , Olson Jeffrey J. , Zhang Zhaobin , Van Meir Erwin G.
Published: September 2005

Hypoxia plays a critical role in driving tumor malignancy and is associated with poor patient survival in many human cancers. Novel therapies targeting hypoxic tumor cells are urgently needed, because these cells hinder tumor eradication. Here we demonstrate than an anticancer strategy based on intratumoral delivery of a novel type of oncolytic adenovirus targeting tumor hypoxia is therapeutically efficient and can augment standard chemotherapy. We used a conditionally replicative adenovirus (HYPR-Ad) to specifically kill hypoxic tumor cells. Viral infection and conditional replication occurred efficiently in hypoxic/hypoxia-inducible factor-active cells in culture and in vivo, prevented tumor formation, and reduced the growth of established tumors. Combining HYPR-Ad with chemotherapy effective against normoxic cells resulted in strongly enhanced antitumor efficacy. These studies demonstrate that targeting the hypoxic microenvironment of tumors rather than an intrinsic gene expression defect is a viable and novel antitumor therapeutic strategy that can be used in combination with existing treatment regimens. The replication and oncolytic potential of this virus was made dependent on hypoxic/hypoxia-inducible factor, a transcription factor activated in the tumor hypoxic microenvironment, broadening its therapeutic use to solid tumors of any genetic make-up or tissue of origin.
Oral Silibinin Inhibits Lung Tumor Growth in Athymic Nude Mice and Forms a Novel Chemocombination with Doxorubicin Targeting Nuclear Factor κB–Mediated Inducible Chemoresistance
Authors: Singh Rana P. , Mallikarjuna G. U. , Sharma Girish , Dhanalakshmi Sivanandhan , Tyagi Anil K. , Chan Daniel C. F. , Agarwal Chapla , Agarwal Rajesh
Published: September 2005

The acute and cumulative dose-related toxicity and drug resistance, mediated via nuclear factor κB (NFκB), of anthracycline anticancer drugs pose a major problem in cancer chemotherapy. Here, we report that oral silibinin (a flavanone) suppresses human non–small-cell lung carcinoma A549 xenograft growth (P = 0.003) and enhances the therapeutic response (P < 0.05) of doxorubicin in athymic BALB/c nu/nu mice together with a strong prevention of doxorubicin-caused adverse health effects. Immunohistochemical analyses of tumors showed that silibinin and doxorubicin decrease (P < 0.001) proliferation index and vasculature and increase (P < 0.001) apoptosis; these effects were further enhanced (P < 0.001) in combination treatment. Pharmacologic dose of silibinin (60 μmol/L) achieved in animal study was biologically effective (P < 0.01 to 0.001, growth inhibition and apoptosis) in vitro in A549 cell culture together with an increased efficacy (P < 0.05 to 0.001) in doxorubicin (25 nmol/L) combination. Furthermore, doxorubicin increased NFκB DNA binding activity as one of the possible mechanisms for chemoresistance in A549 cells, which was inhibited by silibinin in combination treatment. Consistent with this, silibinin inhibited doxorubicin-caused increased translocation of p65 and p50 from cytosol to nucleus. Silibinin also inhibited cyclooxygenase-2, an NFκB target, in doxorubicin combination. These findings suggest that silibinin inhibits in vivo lung tumor growth and reduces systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin-induced chemoresistance involving NFκB signaling.
p16INK4A Hypermethylation Is Associated with Hepatitis Virus Infection, Age, and Gender in Hepatocellular Carcinoma
Authors: Li Xin , Hui Ai-Min , Sun Lin , Hasegawa Kiyoshi , Torzilli Guido , Minagawa Masami , Takayama Tadatoshi , Makuuchi Masatoshi
Published: September 2005

Purpose: The tumor suppressor gene p16INK4A is mainly inactivated by an epigenetic change involving promoter hypermethylation in hepatocarcinogenesis. The possible clinical impact of p16INK4A methylation and the potential risk factors for this epigenetic alteration have not been thoroughly investigated.

Experimental Design: We studied the methylation status and mRNA and protein expression of p16INK4A in 50 hepatocellular carcinomas and corresponding nonneoplastic liver lesions using methylation-specific PCR, reverse transcription-PCR, and immunohistochemical techniques.

Results: p16INK4A hypermethylation was observed in 58% (29 of 50) of the hepatocellular carcinomas and 16% (6 of 38) of the corresponding chronic hepatitis and cirrhosis tissue samples. p16INK4A methylation was significantly associated with mRNA and protein expression (P < 0.001 and P = 0.003, respectively). All of the p16INK4A-methylated tumors were positive for hepatitis B virus or hepatitis C virus markers, but none of the virus-negative tumors exhibited p16INK4A methylation (P = 0.006). The frequency of p16INK4A hypermethylation tended to be higher in hepatitis C virus-related tumors (23 of 32, 72%) than in hepatitis B virus-related tumors (6 of 13, 46%; P = 0.1). Aberrant methylation of p16INK4A was also related significantly to increasing age, female gender, and normal levels of serum PIVKA-II (P = 0.02, 0.04, and 0.04, respectively). No statistically significant difference in survival was observed between patients with p16INK4A hypermethylation and those without.

Conclusions: Our observations suggest that p16INK4A hypermethylation may contribute to hepatocarcinogenesis from an early stage and that multiple risk factors, such as viral infections, age, and gender, may be associated with p16INK4A hypermethylation in hepatocarcinogenesis.
Bovine lactoferricin selectively induces apoptosis in human leukemia and carcinoma cell lines
Authors: Mader Jamie S. , Salsman Jayme , Conrad David M. , Hoskin David W.
Published: September 2005

Bovine lactoferricin (LfcinB) is a cationic, amphipathic peptide that is cytotoxic for human and rodent cancer cells. However, the mechanism by which LfcinB causes the death of cancer cells is not well understood. Here, we show that in vitro treatment with LfcinB rapidly induced apoptosis in several different human leukemia and carcinoma cell lines as determined by DNA fragmentation assays and phosphatidylserine headgroup inversion detected by Annexin V binding to the surface of cancer cells. Importantly, LfcinB treatment did not adversely affect the viability of untransformed human lymphocytes, fibroblasts, or endothelial cells. Studies with different LfcinB-derived peptide fragments revealed that the cytotoxic activity of LfcinB resided within the amino acid sequence FKCRRWQWRM. Treatment of Jurkat T leukemia cells with LfcinB resulted in the production of reactive oxygen species followed by caspase-2-induced dissipation of mitochondrial transmembrane potential and subsequent activation of caspase-9 and caspase-3. Selective inhibitors of caspase-2 (Z-VDVAD-FMK), caspase-9 (Z-LEHD-FMK), and caspase-3 (Z-DEVD-FMK) protected both leukemia and carcinoma cells from LfcinB-induced apoptosis. Conversely, a caspase-8 inhibitor (Z-IETD-FMK) had no effect, which argued against a role for caspase-8 and was consistent with the finding that death receptors were not involved in LfcinB-induced apoptosis. Furthermore, Jurkat T leukemia cells that overexpressed Bcl-2 were less sensitive to LfcinB-induced apoptosis, which was characterized by mitochondrial swelling and the release of cytochrome c from mitochondria into the cytosolic compartment. We conclude that LfcinB kills cancer cells by triggering the mitochondrial pathway of apoptosis at least in part through the generation of reactive oxygen species.
N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy
Authors: Christiansen Jason J. , Rajasekaran Sigrid A. , Inge Landon , Cheng Lirong , Anilkumar Gopalakrishnapillai , Bander Neil H. , Rajasekaran Ayyappan K.
Published: September 2005

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.
Histone deacetylase inhibitors induced caspase-independent apoptosis in human pancreatic adenocarcinoma cell lines
Authors: García-Morales Pilar , Gómez-Martínez Angeles , Carrato Alfredo , Martínez-Lacaci Isabel , Barberá Víctor M. , Soto José L. , Carrasco-García Estefanía , Menéndez-Gutierrez María P. , Castro-Galache María D. , Ferragut José A. , Saceda Miguel
Published: September 2005

The antitumor activity of the histone deacetylase inhibitors was tested in three well-characterized pancreatic adenocarcinoma cell lines, IMIM-PC-1, IMIM-PC-2, and RWP-1. These cell lines have been previously characterized in terms of their origin, the status of relevant molecular markers for this kind of tumor, resistance to other antineoplastic drugs, and expression of differentiation markers. In this study, we report that histone deacetylase inhibitors induce apoptosis in pancreatic cancer cell lines, independently of their intrinsic resistance to conventional antineoplastic agents. The histone deacetylase inhibitor–induced apoptosis is due to a serine protease–dependent and caspase-independent mechanism. Initially, histone deacetylase inhibitors increase Bax protein levels without affecting Bcl-2 levels. Consequently, the apoptosis-inducing factor (AIF) and Omi/HtrA2 are released from the mitochondria, with the subsequent induction of the apoptotic program. These phenomena require AIF relocalization into the nuclei to induce DNA fragmentation and a serine protease activity of Omi/HtrA2. These data, together with previous results from other cellular models bearing the multidrug resistance phenotype, suggest a possible role of the histone deacetylase inhibitors as antineoplastic agents for the treatment of human pancreatic adenocarcinoma.
p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome
Authors: Nakase Minoru , Inui Madoka , Okumura Kenya , Kamei Takahiko , Nakamura Shinnosuke , Tagawa Toshiro
Published: September 2005

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.
Inhibition of the epidermal growth factor receptor increases expression of genes that stimulate inflammation, apoptosis, and cell attachment
Authors: Woodworth Craig D. , Michael Evan , Marker Dan , Allen Sarah , Smith Laura , Nees Matthias
Published: September 2005

The epidermal growth factor receptor (EGFR) is overexpressed in several types of human cancer, and inhibition of EGFR function is a promising strategy for cancer therapy. We used cDNA microarrays to examine alterations in gene expression after treatment of carcinoma cells with PD153035, a specific and reversible inhibitor of EGFR function. When human cervical carcinoma cells were grown on a collagen substrate in three-dimensional organotypic culture, untreated cells expressed high levels of EGFR RNA and invaded the underlying collagen. Blocking EGFR function decreased DNA synthesis and inhibited invasion in a dose-dependent manner. Microarray analyses identified 312 genes that were significantly increased or decreased in expression after EGFR inhibition. Many could be classified into one of four functional groups including genes that (a) stimulate inflammation and innate immunity, (b) promote cell attachment, (c) enhance apoptosis, and (d) inhibit cell cycle progression. PD153035 induced a dose-dependent activation of nuclear factor κB, a transcription factor that stimulates proinflammatory gene expression. Our results identify alterations in gene expression caused by EGFR inhibition and show that this response varies significantly in different cell lines.
Human mesothelioma cells exhibit tumor cell–specific differences in phosphatidylinositol 3-kinase/AKT activity that predict the efficacy of Onconase
Authors: Ramos-Nino Maria E. , Vianale Giovina , Sabo-Attwood Tara , Mutti Luciano , Porta Camilo , Heintz Nicholas , Mossman Brooke T.
Published: September 2005

Malignant mesothelioma is an aggressive cancer with no known cure, which has become a therapeutic challenge. Onconase is one of few chemotherapeutic agents that have been studied in patients with malignant mesothelioma that has the advantage of low toxicity and limited side effects. Here, we evaluate the effect of Onconase on killing of malignant mesothelioma cells and how the phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) survival pathway influences this effect. Our results show that Onconase induces apoptosis in malignant mesothelioma cell lines and that this effect is tumor cell specific. Malignant mesothelioma cell lines with the highest AKT activation, which correlated with the presence of the SV40 large and small T antigen (SV40+), were the most resistant to the drug. Finally, a cooperative effect was observed between small molecule inhibitors of PI3-K and Onconase in the killing of malignant mesothelioma cells. Our results suggest that kinase screening of individual malignant mesotheliomas for endogenous levels of activated PI3-K/AKT may be predictive of the efficacy of Onconase and possibly other chemotherapeutic agents.
Novel hydroxyl naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties
Authors: Peyregne Vincent P. , Kar Siddhartha , Ham Seung W. , Wang Meifang , Wang Ziqiu , Carr Brian I.
Published: September 2005

Cdc25 phosphatases are important in cell cycle control and activate cyclin-dependent kinases (Cdk). Efforts are currently under way to synthesize specific small-molecule Cdc25 inhibitors that might have anticancer properties. NSC 95397, a protein tyrosine phosphatase antagonist from the National Cancer Institute library, was reported to be a potent Cdc25 inhibitor. We have synthesized two hydroxyl derivatives of NSC 95397, monohydroxyl-NSC 95397 and dihydroxyl-NSC 95397, which both have enhanced activity for inhibiting Cdc25s. The new analogues, especially dihydroxyl-NSC 95397, potently inhibited the growth of human hepatoma and breast cancer cells in vitro. They influenced two signaling pathways. The dual phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was induced, likely due to inhibition of the ERK phosphatase activity in Hep 3B cell lysate but not the dual specificity ERK phosphatase MKP-1. They also inhibited Cdc25 enzymatic activities and induced tyrosine phosphorylation of the Cdc25 target Cdks. Addition of hydroxyl groups to the naphthoquinone ring thus enhanced the potency of NSC 95397. These two new compounds may be useful probes for the biological functions of Cdc25s and have the potential for disrupting the cell cycle of growing tumor cells.
Oridonin, a diterpenoid purified fromRabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-κB signal pathways
Authors: Ikezoe Takayuki , Yang Yang , Bandobashi Kentaro , Saito Tsuyako , Takemoto Shigeki , Machida Hisanori , Togitani Kazuto , Koeffler H. Phillip , Taguchi Hirokuni
Published: September 2005

This study found that oridonin, a natural diterpenoid purified from Rabdosia rubescens, inhibited growth of multiple myeloma (MM; U266, RPMI8226), acute lymphoblastic T-cell leukemia (Jurkat), and adult T-cell leukemia (MT-1) cells with an effective dose that inhibited 50% of target cells (ED50) ranging from 0.75 to 2.7 μg/mL. Terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling staining showed that oridonin caused apoptosis of MT-1 cells in a time-dependent manner. We explored effects of oridonin on antiapoptotic Bcl-2 family members and found that it down-regulated levels of Mcl-1 and BCL-xL, but not Bcl-2 protein, in both MT-1 and RPMI8226 cells. Further studies found that oridonin inhibited nuclear factor-κB (NF-κB) DNA-binding activity in these cells as measured by luciferase reporter gene, ELISA-based, and electrophoretic mobility shift assays. Oridonin also blocked tumor necrosis factor-α– and lipopolysaccharide-stimulated NF-κB activity in Jurkat cells as well as RAW264.7 murine macrophages. Of note, oridonin decreased survival of freshly isolated adult T-cell leukemia (three samples), acute lymphoblastic leukemia (one sample), chronic lymphocytic leukemia (one sample), non-Hodgkin's lymphoma (three samples), and MM (four samples) cells from patients in association with inhibition of NF-κB DNA-binding activity. On the other hand, oridonin did not affect survival of normal lymphoid cells from healthy volunteers. Taken together, oridonin might be useful as adjunctive therapy for individuals with lymphoid malignancies, including the lethal disease adult T-cell leukemia.
Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824
Authors: Chen Liwei , Meng Songshu , Wang Hai , Bali Purva , Bai Wenlong , Li Benyi , Atadja Peter , Bhalla Kapil N. , Wu Jie
Published: September 2005

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.
Discovery and preclinical evaluation of a novel class of small-molecule compounds in hormone-dependent and -independent cancer cell lines
Authors: Plasencia Carmen , Dayam Raveendra , Wang Qingcai , Pinski Jacek , Burke Terrence R. , Quinn David I. , Neamati Nouri
Published: September 2005

We discovered a series of salicylhydrazide class of compounds with remarkable anticancer activity against a panel of hormone receptor–positive and -negative cell lines. In the present study, we evaluated the in vitro activity of SC21 and SC23 against a range of human tumor cell types and the in vivo efficacy of compound SC21 in a PC3 human prostate cancer xenograft model in mice. We also determined the effects of SC21 on cell cycle regulation and apoptosis. Our in vitro results show that salicylhydrazides are highly potent compounds effective in both hormone receptor–positive and -negative cancer cells. SC21 induced apoptosis and blocked the cell cycle in G0/G1 or S phase, depending on the cell lines used and irrespective of p53, p21, pRb, and p16 status. SC21 effectively reduced the tumor growth in mice without apparent toxicity. Although the mechanism of action of SC21 is not completely elucidated, the effect on cell cycle, the induction of apoptosis and the activity against a panel of tumor cell lines of different origins prompted us to carry out an in-depth preclinical evaluation of SC21.
The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites
Authors: Pratt Susan , Shepard Robert L. , Kandasamy Ramani A. , Johnston Paul A. , Perry William , Dantzig Anne H.
Published: September 2005

5′-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5′-fluoro-2′-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5′-fluoro-2′-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were ∼9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 μmol/L [3H]5-FdUMP, [3H]5-FUMP, [3H]dUMP, and not [3H]5-FUdR, or [3H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a Km of 1.1 mmol/L and Vmax of 439 pmol/min/mg protein. Uptake of 250 μmol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3′,5′-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 μmol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.
8-Amino-adenosine induces loss of phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt kinase: Role in induction of apoptosis in multiple myeloma
Authors: Ghias Kulsoom , Ma Chunguang , Gandhi Varsha , Platanias Leonidas C. , Krett Nancy L. , Rosen Steven T.
Published: September 2005

Multiple myeloma is a slowly proliferating B-cell malignancy that accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics that target rapidly replicating cells. Multiple myeloma remains an incurable disease in need of new therapeutic approaches. The purine nucleoside analogue, 8-amino-adenosine (8-NH2-Ado), exhibits potent activity in preclinical studies, inducing apoptosis in several multiple myeloma cell lines. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its triphosphate form, 8-amino-ATP, and results in a concomitant loss of endogenous ATP levels. Here, we show the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. Multiple myeloma cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and Akt kinase. Cells depleted of ATP independent of 8-NH2-Ado do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may influence the activity of select regulatory protein kinases and/or phosphatases, with preliminary data suggesting that protein phophatase 2A activity is affected by 8-NH2-Ado. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analogue drug and is unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical prosurvival and apoptosis-regulating proteins suggests that the modulation of these proteins by dephosphorylation at early time points may be an important mechanistic step in 8-NH2-Ado-induced apoptosis.
Strongylophorine-26, a Rho-dependent inhibitor of tumor cell invasion that reduces actin stress fibers and induces nonpolarized lamellipodial extensions
Authors: McHardy Lianne M. , Warabi Kaoru , Andersen Raymond J. , Roskelley Calvin D. , Roberge Michel
Published: September 2005

Strongylophorine-26, a new meroditerpenoid, was recently identified as an inhibitor of cancer cell invasion. This study was undertaken to characterize its mechanism of action. We find that strongylophorine-26 inhibits the motility of MDA-MB-231 breast carcinoma cells on a plastic surface. Upon addition of strongylophorine-26, rapid cell contraction and depolarization occurred, followed by spreading and flattening of the entire cell. Treated cells exhibited increased membrane ruffling throughout and extended lamellipodia in all directions. Strongylophorine-26 induced a decrease in actin stress fibers, a dramatic increase in the size and number of focal adhesions, and the appearance of a dense meshwork of actin filaments around the cell periphery. Strongylophorine-26 caused a transient activation of the small GTPase Rho and treatment with the Rho inhibitor C3 exoenzyme abrogated the anti-invasive activity of strongylophorine-26. These effects are distinct from those of many motility and angiogenesis inhibitors that seem to act by a common mechanism involving the induction of actin stress fibers. This difference in mechanism of action sets strongylophorine-26 apart as an experimental anticancer agent and indicates that pharmacologic inhibition of cell migration may be achieved by mechanisms not involving the stabilization of actin stress fibers.
Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein
Authors: Cho Hyun-Mi , Rosenblatt Joseph D. , Kang Young-Sook , Iruela-Arispe M. Luisa , Morrison Sherie L. , Penichet Manuel L. , Kwon Young-Guen , Kim Tae-Woong , Webster Keith A. , Nechustan Hovav , Shin Seung-Uon
Published: September 2005

Endostatin can inhibit angiogenesis and tumor growth in mice. A potential limitation of endostatin as an antitumor agent in humans is the short serum half-life of the protein that may decrease effective concentration at the site of tumor and necessitate frequent dosing. In an effort to improve antitumor activity, endostatin was fused to an antibody specific for the tumor-selective HER2 antigen to create an antibody-endostatin fusion protein (anti-HER2 IgG3-endostatin). Normal endostatin rapidly cleared from serum in mice (T1/22, = 0.6–3.8 hours), whereas anti-HER2 IgG3-endostatin had a prolonged half-life (90% intact; T1/22, 40.2–44.0 hours). Antigen-specific targeting of anti-HER2 IgG3-endostatin was evaluated in BALB/c mice implanted with CT26 tumors or CT26 tumors engineered to express the HER2 antigen (CT26-HER2). Radio-iodinated anti-HER2 IgG3-endostatin preferentially localized to CT26-HER2 tumors relative to CT26 tumors. Administration of anti-HER2 IgG3-endostatin to mice showed preferential inhibition of CT26-HER2 tumor growth compared with CT26. Anti-HER2 IgG3-endostatin also markedly inhibited the growth of human breast cancer SK-BR-3 xenografts in severe combined immunodeficient mice. Anti-HER2 IgG3-endostatin inhibited tumor growth significantly more effectively than endostatin, anti-HER2 IgG3 antibody, or the combination of antibody and endostatin. CT26-HER2 tumors treated with the endostatin fusion protein had decreased blood vessel density and branching compared with untreated CT26-HER2 or CT26 treated with the fusion protein. The enhanced effectiveness of anti-HER2 IgG3-endostatin may be due to a longer half-life, improved serum stability, and selective targeting of endostatin to tumors, resulting in decreased angiogenesis. Linking of an antiangiogenic protein, such as endostatin, to a targeting antibody represents a promising and versatile approach to antitumor therapy.
Manganese superoxide dismutase and inducible nitric oxide synthase modify early oxidative events in acute Adriamycin-induced mitochondrial toxicity
Authors: Chaiswing Luksana , Cole Marsha P. , Ittarat Wanida , Szweda Luke I. , St. Clair Daret K. , Oberley Terry D.
Published: September 2005

In the present study, we used genetically engineered B6C3 mice [mice overexpressing manganese superoxide dismutase (TgM+/+), mice in which inducible nitric oxide synthase had been inactivated (iNOSKO−/−), and crosses of these two genotypes] to study the role of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in the development of acute Adriamycin-induced cardiotoxicity. Both nontransgenic and genetically engineered mice were treated with 20 mg/kg Adriamycin and cardiac left ventricular tissues studied at 0, 3, 6, and 24 hours. Ultrastructural damage and levels of 4-hydroxy-2-nonenal (4HNE) protein adducts and 3-nitrotyrosine (3NT) were determined in cardiomyocytes using immunogold ultrastructural techniques. Our previous results showed that Adriamycin caused mitochondrial injury without significant nuclear or cytoplasmic damage at early time points. Interestingly, overexpression of MnSOD protected against acute mitochondrial injury, whereas deficiency in iNOS potentiated mitochondrial injury in comparison with levels of injury present in cardiomyocyte mitochondria of nontransgenic mice. In TgM+/+ mice, there was a significant inverse correlation between mitochondrial injury and 4HNE/3NT levels at all time points analyzed, suggesting that reactive oxygen species/reactive nitrogen species damage products directly regulated acute Adriamycin-induced mitochondrial injury in these mice. The present studies are the first to directly quantify the effects of MnSOD and iNOS on mitochondrial injury during acute Adriamycin-induced cardiotoxicity and show extensive and specific patterns of posttranslational modifications of mitochondrial proteins following Adriamycin treatment.
The discovery and mechanism of action of novel tumor-selective and apoptosis-inducing 3,5-diaryl-1,2,4-oxadiazole series using a chemical genetics approach
Authors: Jessen Katayoun A. , English Nicole M. , Yu Wang Jean , Maliartchouk Sergei , Archer Shannon P. , Qiu Ling , Brand Regina , Kuemmerle Jared , Zhang Han-Zhong , Gehlsen Kurt , Drewe John , Tseng Ben , Cai Sui Xiong , Kasibhatla Shailaja
Published: September 2005

A novel series of 3,5-diaryl-oxadiazoles was identified as apoptosis-inducing agents through our cell and chemical genetics–based screening assay for compounds that induce apoptosis using a chemical genetics approach. Several analogues from this series including MX-74420 and MX-126374 were further characterized. MX-126374, a lead compound from this series, was shown to induce apoptosis and inhibit cell growth selectively in tumor cells. To elucidate the mechanism(s) by which this class of compounds alters the signal transduction pathway that ultimately leads to apoptosis, expression profiling using the Affymetrix Gene Chip array technology was done along with other molecular and biochemical analyses. Interestingly, we have identified several key genes (cyclin D1, transforming growth factor-β1, p21, and insulin-like growth factor-BP3) that are altered in the presence of this compound, leading to characterization of the pathway for activation of apoptosis. MX-126374 also showed significant inhibition of tumor growth as a single agent and in combination with paclitaxel in murine tumor models. Using photoaffinity labeling, tail-interacting protein 47, an insulin-like growth factor-II receptor binding protein, was identified as the molecular target. Further studies indicated that down-regulation of tail-interacting protein 47 in cancer cells by small interfering RNA shows a similar pathway profile as compound treatment. These data suggest that 3,5-diaryl-oxadiazoles may be a new class of anticancer drugs that are tumor-selective and further support the discovery of novel drugs and drug targets using chemical genetic approaches.
The anti-invasive activity of cyclooxygenase inhibitors is regulated by the transcription factor ATF3 (activating transcription factor 3)
Authors: Bottone Frank G. , Moon Yuseok , Kim Jong Sik , Alston-Mills Brenda , Ishibashi Minako , Eling Thomas E.
Published: September 2005

We previously showed that nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide, which has chemopreventive activity, modulate the expression of several genes detected by microarray analysis. Activating transcription factor 3 (ATF3) was selected for further study because it is a transcription factor involved in cell proliferation, apoptosis, and invasion, and its expression is repressed in human colorectal tumors as compared with normal adjacent tissue. In this report, we show that ATF3 mRNA and protein expression are up-regulated in HCT-116 human colorectal cancer cells following treatment with NSAIDs, troglitazone, diallyl disulfide, and resveratrol. To ascertain the biological significance of ATF3, we overexpressed full-length ATF3 protein in the sense and antisense orientations. Overexpression of ATF3 in the sense orientation decreased focus formation in vitro and reduced the size of mouse tumor xenografts by 54% in vivo. Conversely, overexpression of antisense ATF3 was protumorigenic in vitro, however, not in vivo. ATF3 in the sense orientation did not modulate apoptosis, indicating another mechanism is involved. With microarray analysis, several genes relating to invasion and metastasis were identified by ATF3 overexpression and were confirmed by real-time reverse transcription-PCR, and several of these genes were modulated by sulindac sulfide, which inhibited invasion in these cells. Furthermore, overexpression of ATF3 inhibited invasion to a similar degree as sulindac sulfide treatment, whereas antisense ATF3 increased invasion. In conclusion, ATF3 represents a novel mechanism in which NSAIDs exert their anti-invasive activity, thereby linking ATF3 and its gene regulatory activity to the biological activity of these compounds.
In vitrocytotoxicity of carcinoma cells with 111In-labeled antibodies to HER-2
Authors: Michel Rosana B. , Andrews Philip M. , Castillo Mary Ellen , Mattes M. Jules
Published: September 2005

Antibodies conjugated to radionuclides emitting low-energy electrons, which include Auger electrons and some conversion electrons, were recently shown to efficiently kill cells bearing a high density of the antigen recognized. The primary purpose of this study was to determine if such killing could be obtained with anti–HER-2 antibodies conjugated to 111In, using the chelator benzyl-diethylenetriaminepentaacetic acid, or 125I. Target cells were the breast carcinoma SK-BR-3 and the ovarian carcinoma SK-OV-3.ip1. In preliminary experiments, antibody accumulation and catabolism during a 2- to 3-day incubation with antibody was investigated. The level of antibody uptake, in terms of molecules per cell, was high enough such that killing seemed feasible. With an 125I label, but not an 111In label, increasing the antibody concentration past a certain point caused a decrease in total antibody accumulation, which might be attributed to effects of antibody binding. To test for cytotoxicity, cells were incubated for 2 days with the labeled antibody, then assayed for colony-forming units with a limiting dilution assay. SK-BR-3 cells were strongly killed (∼3 logs) by antibody 21.1, and 100% kill was obtained by combining two noncompeting antibodies to HER-2 (21.1 and 4D5). SK-OV-3.ip.1 cells were more resistant to killing, but use of the two-antibody mixture produced a surviving fraction of ∼0.002. 111In-labeled antibodies to other high-density antigens, epithelial glycoprotein-1 and epithelial glycoprotein-2, also killed these target cells. In contrast, unlabeled antibodies or a nonreactive-labeled antibody produced much less cytotoxicity. The same experiment with an 131I label (a β-particle emitter) resulted in much greater levels of nonspecific cytotoxicity and essentially no specific cytotoxicity. This approach may be effective for therapy of micrometastases.
p53 α-Helix mimetics antagonize p53/MDM2 interaction and activate p53
Authors: Chen Lihong , Yin Hang , Farooqi Bilal , Sebti Said , Hamilton Andrew D. , Chen Jiandong
Published: September 2005

Overexpression or hyperactivation of MDM2 contributes to functional inactivation of wild-type p53 in nearly 50% of tumors. Inhibition of p53 by MDM2 depends on binding between an NH2-terminal (residues 16–28) p53 α-helical peptide and a hydrophobic pocket on MDM2, presenting an attractive target for development of inhibitors against tumors expressing wild-type p53. Here we report that novel p53 α-helical peptide mimics based on a terphenyl scaffold can inhibit MDM2-p53 binding in vitro and activate p53 in vivo. Several active compounds have been identified that inhibit MDM2-p53 binding in an ELISA assay with IC50 of 10 to 20 μmol/L and induce p53 accumulation and activation in cell culture at 15 to 40 μmol/L. These results suggest that p53 α-helical mimetics based on the terphenyl scaffold may be developed into potent p53 activators.
Farnesyltransferase inhibitors reverse altered growth and distribution of actin filaments inTsc-deficient cells via inhibition of both rapamycin-sensitive and -insensitive pathways
Authors: Gau Chia-Ling , Kato-Stankiewicz Juran , Jiang Chen , Miyamoto Susie , Guo Lea , Tamanoi Fuyuhiko
Published: September 2005

Farnesyltransferase inhibitors (FTI) have been developed as anticancer drugs and are currently being evaluated in clinical trials. In this study, we have examined the effects of FTIs on Tsc-null cells to gain insight into their effects on farnesylated Rheb GTPase. This protein is involved in the activation of mTOR/S6K signaling and is down-regulated by the Tsc1/Tsc2 complex. Both Tsc1−/− and Tsc2−/− mouse embryonic fibroblasts exhibit constitutive activation of S6K and grow in the absence of serum. Two different FTI compounds, the clinical compound BMS-214662 and the newly described BMS-225975, inhibit the constitutive activation of mTOR/S6K signaling and block serum-free growth of the Tsc-null mouse embryonic fibroblasts. We have also found that Tsc-null mouse embryonic fibroblasts grow under anchorage-independent conditions and that both FTI compounds inhibit this soft agar growth. These FTI effects are similar to those observed with rapamycin. Another interesting phenotype of Tsc-null mouse embryonic fibroblasts is that they are round and contain actin filaments predominantly at the cell periphery. The addition of FTIs, but not rapamycin, led to the reappearance of intracellular actin filaments and reduction of peripheral actin filaments. The ability of FTI to rearrange actin filaments seems to be largely mediated by the inhibition of Rheb protein, as induction of intracellular actin filaments by FTI was much less efficient in Tsc2-null cells expressing Rheb (M184L), a geranylgeranylated mutant Rheb that can bypass farnesylation. These results reveal that FTIs inhibit Rheb, causing two different effects in Tsc-deficient cells, one on growth and the other on actin filament distribution.
Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis
Authors: D'Andrea Michael R. , Mei Jay M. , Tuman Robert W. , Galemmo Robert A. , Johnson Dana L.
Published: September 2005

With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cγ1 (PLCγ1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCγ1 (pan-PLCγ1) and the other, specific for phosphorylated form of PLCγ1 (ph-PLCγ1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCγ1 to pan-PLCγ1 immunolabeling intensities in serial sections (5 μm) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCγ as a downstream signaling partner, these methods may also benefit other drug discovery targets.
Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo
Authors: Luo Yan , Shoemaker Alexander R. , Liu Xuesong , Woods Keith W. , Thomas Sheela A. , de Jong Ron , Han Edward K. , Li Tongmei , Stoll Vincent S. , Powlas Jessica A. , Oleksijew Anatol , Mitten Michael J. , Shi Yan , Guan Ran , McGonigal Thomas P. , Klinghofer Vered , Johnson Eric F. , Leverson Joel D. , Bouska Jennifer J. , Mamo Mulugeta , Smith Richard A. , Gramling-Evans Emily E. , Zinker Bradley A. , Mika Amanda K. , Nguyen Phong T. , Oltersdorf Tilman , Rosenberg Saul H. , Li Qun , Giranda Vincent L.
Published: September 2005

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (Ki = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses ∼2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.
Selective and irreversible cell cycle inhibition by diphenyleneiodonium
Authors: Scaife Robin M.
Published: September 2005

Because cell proliferation is subject to checkpoint-mediated regulation of the cell cycle, pharmacophores that target cell cycle checkpoints have been used clinically to treat human hyperproliferative disorders. It is shown here that the flavoprotein inhibitor diphenyleneiodionium can block cell proliferation by targeting of cell cycle checkpoints. Brief exposure of mitotically arrested cells to diphenyleneiodonium induces a loss of the mitotic cell morphology, and this corresponds with a decrease in the levels of the mitotic markers MPM2 and phospho-histone H3, as well as a loss of centrosome maturation, spindle disassembly, and redistribution of the chromatin remodeling helicase ATRX. Surprisingly, this mitotic exit resulted in a tetraploidization that persisted long after drug release. Analogously, brief exposure to diphenyleneiodonium also caused prolonged arrest in G1 phase. By contrast, diphenyleneiodonium exposure did not abrogate S phase, although it did result in a subsequent block of G2 cell cycle progression. This indicates that diphenyleneiodonium selectively targets components of the cell cycle, thereby either causing cell cycle arrest, or checkpoint override followed by cell cycle arrest. These irreversible effects of diphenyleneiodonium on the cell cycle may underlie its potent antiproliferative activity.